Journal articles: 'Specific protein/RNA recognition'

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Stolarski, Ryszard. "Thermodynamics of specific protein-RNA interactions." Acta Biochimica Polonica 50, no. 2 (June 30, 2003): 297–318. http://dx.doi.org/10.18388/abp.2003_3688.

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Description of the recognition specificity between proteins and nucleic acids at the level of molecular interactions is one of the most challenging tasks in biophysics. It is key to understanding the course and control of gene expression and to the application of the thus acquired knowledge in chemotherapy. This review presents experimental results of thermodynamic studies and a discussion of the role of thermodynamics in formation and stability of functional protein-RNA complexes, with a special attention to the interactions involving mRNA 5' cap and cap-binding proteins in the initiation of protein biosynthesis in the eukaryotic cell. A theoretical framework for analysis of the thermodynamic parameters of protein-nucleic acid association is also briefly surveyed. Overshadowed by more spectacular achievements in structural studies, the thermodynamic investigations are of equal importance for full comprehension of biopolymers' activity in a quantitative way. In this regard, thermodynamics gives a direct insight into the energetic and entropic characteristics of complex macromolecular systems in their natural environment, aqueous solution, and thus complements the structural view derived from X-ray crystallography and multidimensional NMR. Further development of the thermodynamic approach toward interpretation of recognition and binding specificity in terms of molecular biophysics requires more profound contribution from statistical mechanics.

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Shen, Cuicui, Xiang Wang, Yexing Liu, Quanxiu Li, Zhao Yang, Nieng Yan, Tingting Zou, and Ping Yin. "Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein." Molecular Plant 8, no. 4 (April 2015): 667–70. http://dx.doi.org/10.1016/j.molp.2015.01.001.

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Qian, Kaiyue, Mengyu Li, Junchao Wang, Min Zhang, and Mingzhu Wang. "Structural basis for mRNA recognition by human RBM38." Biochemical Journal 477, no. 1 (January 10, 2020): 161–72. http://dx.doi.org/10.1042/bcj20190652.

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RNA-binding protein RBM38 was reported to bind the mRNA of several p53-related genes through its RRM domain and to up-regulate or down-regulate protein translation by increasing mRNA stability or recruitment of other effector proteins. The recognition mechanism, however, for RNA-binding of RBM38 remains unclear. Here, we report the crystal structure of the RRM domain of human RBM38 in complex with a single-stranded RNA. Our structural and biological results revealed that RBM38 recognizes G(U/C/A)GUG sequence single-stranded RNA in a sequence-specific and structure-specific manner. Two phenylalanine stacked with bases of RNA were crucial for RNA binding, and a series of hydrogen bonds between the base atoms of RNA and main-chain or side-chain atoms of RBM38 determine the sequence-specific recognition. Our results revealed the RNA-recognition mechanism of human RBM38 and provided structural information for understanding the RNA-binding property of RBM38.

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Spiridonova, V. A. "Molecular recognition elements - DNA/RNA-aptamers to proteins." Biomeditsinskaya Khimiya 56, no. 6 (2010): 639–56. http://dx.doi.org/10.18097/pbmc20105606639.

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In this review summarizes data on DNA/RNA aptamers - a novel class of molecular recognition elements. Special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. These include aptamers to serine protease, to cytokines/growth factors, to influenza viral protein, nucleic acid binding proteins. Strong and specific binding for a given protein target of aptamers make them an attractive class of direct protein inhibitors. They can inhibit pathogenic proteins and it is becoming clear that aptamers have the potential to be a new and effective class of therapeutic molecules.

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Pérez-Cano, Laura, and Juan Fernández-Recio. "Dissection and prediction of RNA-binding sites on proteins." BioMolecular Concepts 1, no. 5-6 (December 1, 2010): 345–55. http://dx.doi.org/10.1515/bmc.2010.037.

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AbstractRNA-binding proteins are involved in many important regulatory processes in cells and their study is essential for a complete understanding of living organisms. They show a large variability from both structural and functional points of view. However, several recent studies performed on protein-RNA crystal structures have revealed interesting common properties. RNA-binding sites usually constitute patches of positively charged or polar residues that make most of the specific and non-specific contacts with RNA. Negatively charged or aliphatic residues are less frequent at protein-RNA interfaces, although they can also be found either forming aliphatic and positive-negative pairs in protein RNA-binding sites or contacting RNA through their main chains. Aromatic residues found within these interfaces are usually involved in specific base recognition at RNA single-strand regions. This specific recognition, in combination with structural complementarity, represents the key source for specificity in protein-RNA association. From all this knowledge, a variety of computational methods for prediction of RNA-binding sites have been developed based either on protein sequence or on protein structure. Some reported methods are really successful in the identification of RNA-binding proteins or the prediction of RNA-binding sites. Given the growing interest in the field, all these studies and prediction methods will undoubtedly contribute to the identification and comprehension of protein-RNA interactions.

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Haynes, S. R., M. T. Cooper, S. Pype, and D. T. Stolow. "Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis." Molecular and Cellular Biology 17, no. 5 (May 1997): 2708–15. http://dx.doi.org/10.1128/mcb.17.5.2708.

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RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis.

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Shi, H., B. E. Hoffman, and J. T. Lis. "A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52." Molecular and Cellular Biology 17, no. 5 (May 1997): 2649–57. http://dx.doi.org/10.1128/mcb.17.5.2649.

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B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

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Kress, Tracy L., Young J. Yoon, and Kimberly L. Mowry. "Nuclear RNP complex assembly initiates cytoplasmic RNA localization." Journal of Cell Biology 165, no. 2 (April 19, 2004): 203–11. http://dx.doi.org/10.1083/jcb.200309145.

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Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA–protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.

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Narayanan, Krishna, Chun-Jen Chen, Junko Maeda, and Shinji Makino. "Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal." Journal of Virology 77, no. 5 (March 1, 2003): 2922–27. http://dx.doi.org/10.1128/jvi.77.5.2922-2927.2003.

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ABSTRACT For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-packaging signal interaction led to specific packaging of the packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA packaging mechanism for an enveloped virus, where the specific RNA packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.

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Schneemann, Anette, and Dawn Marshall. "Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha." Journal of Virology 72, no. 11 (November 1, 1998): 8738–46. http://dx.doi.org/10.1128/jvi.72.11.8738-8746.1998.

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ABSTRACT Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.

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McLaughlin, Krystle J., Jermaine L. Jenkins, and Clara L. Kielkopf. "Large Favorable Enthalpy Changes Drive Specific RNA Recognition by RNA Recognition Motif Proteins." Biochemistry 50, no. 9 (March 8, 2011): 1429–31. http://dx.doi.org/10.1021/bi102057m.

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Mir, M. A., B. Brown, B. Hjelle, W. A. Duran, and A. T. Panganiban. "Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle." Journal of Virology 80, no. 22 (September 13, 2006): 11283–92. http://dx.doi.org/10.1128/jvi.00820-06.

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ABSTRACT A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into “panhandle” hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5′ and 3′ ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

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Soffer, Adam, Sarah A. Eisdorfer, Morya Ifrach, Stefan Ilic, Ariel Afek, Hallel Schussheim, Dan Vilenchik, and Barak Akabayov. "Inferring primase-DNA specific recognition using a data driven approach." Nucleic Acids Research 49, no. 20 (October 29, 2021): 11447–58. http://dx.doi.org/10.1093/nar/gkab956.

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Abstract DNA–protein interactions play essential roles in all living cells. Understanding of how features embedded in the DNA sequence affect specific interactions with proteins is both challenging and important, since it may contribute to finding the means to regulate metabolic pathways involving DNA–protein interactions. Using a massive experimental benchmark dataset of binding scores for DNA sequences and a machine learning workflow, we describe the binding to DNA of T7 primase, as a model system for specific DNA–protein interactions. Effective binding of T7 primase to its specific DNA recognition sequences triggers the formation of RNA primers that serve as Okazaki fragment start sites during DNA replication.

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Brown, Rebecca S., Lisa Kim, and Margaret Kielian. "Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein." Viruses 13, no. 8 (July 31, 2021): 1517. http://dx.doi.org/10.3390/v13081517.

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Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

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WOESTENENK, Esmeralda A., George M. GONGADZE, Dmitry V. SHCHERBAKOV, Alexey V. RAK, Maria B. GARBER, Torleif HÄRD, and Helena BERGLUND. "The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold." Biochemical Journal 363, no. 3 (April 24, 2002): 553–61. http://dx.doi.org/10.1042/bj3630553.

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We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5kDa protein of the large subunit of the ribosome and binds to both 5S and 23S rRNA. In the uncomplexed state L18 folds to a mixed α/β globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting β-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA—protein-recognition module is tolerant to variations in sequence.

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Ghosh, Meenakshi, and Mahavir Singh. "Structure specific recognition of telomeric repeats containing RNA by the RGG-box of hnRNPA1." Nucleic Acids Research 48, no. 8 (March 4, 2020): 4492–506. http://dx.doi.org/10.1093/nar/gkaa134.

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Abstract The telomere repeats containing RNA (TERRA) is transcribed from the C-rich strand of telomere DNA and comprises of UUAGGG nucleotides repeats in humans. The TERRA RNA repeats can exist in single stranded, RNA-DNA hybrid and G-quadruplex forms in the cell. Interaction of TERRA RNA with hnRNPA1 has been proposed to play critical roles in maintenance of telomere DNA. hnRNPA1 contains an N-terminal UP1 domain followed by an RGG-box containing C-terminal region. RGG-motifs are emerging as key protein motifs that recognize the higher order nucleic acid structures as well as are known to promote liquid-liquid phase separation of proteins. In this study, we have shown that the RGG-box of hnRNPA1 specifically recognizes the TERRA RNA G-quadruplexes that have loops in their topology, whereas it does not interact with the single-stranded RNA. Our results show that the N-terminal UP1 domain in the presence of the RGG-box destabilizes the loop containing TERRA RNA G-quadruplex efficiently compared to the RNA G-quadruplex that lacks loops, suggesting that unfolding of G-quadruplex structures by UP1 is structure dependent. Furthermore, we have compared the telomere DNA and TERRA RNA G-quadruplex binding by the RGG-box of hnRNPA1 and discussed its implications in telomere DNA maintenance.

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Nagarajan, Raju, Sonia Chothani, Chandrasekaran Ramakrishnan, Masakazu Sekijima, and M. Gromiha. "Structure based approach for understanding organism specific recognition of protein-RNA complexes." Biology Direct 10, no. 1 (2015): 8. http://dx.doi.org/10.1186/s13062-015-0039-8.

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Laurenzi, Tommaso, Luca Palazzolo, Elisa Taiana, Simona Saporiti, Omar Ben Mariem, Uliano Guerrini, Antonino Neri, and Ivano Eberini. "Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism." International Journal of Molecular Sciences 23, no. 14 (July 10, 2022): 7626. http://dx.doi.org/10.3390/ijms23147626.

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NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein–protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer’s RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

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Zhao, Yongqian, Tingjin Sherryl Soh, Siew Pheng Lim, Ka Yan Chung, Kunchithapadam Swaminathan, Subhash G. Vasudevan, Pei-Yong Shi, Julien Lescar, and Dahai Luo. "Molecular basis for specific viral RNA recognition and 2′-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5)." Proceedings of the National Academy of Sciences 112, no. 48 (November 17, 2015): 14834–39. http://dx.doi.org/10.1073/pnas.1514978112.

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Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5′ exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5′ RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5′ RNA cap: G0pppAG-RNA→m7G0pppAG-RNA (“cap-0”)→m7G0pppAm2′-O-G-RNA (“cap-1”). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5′-m7G0pppA1G2U3U4G5U6U7-3′), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2′-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.

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Vasilyev, Nikita, Anna Polonskaia, Jennifer C. Darnell, Robert B. Darnell, Dinshaw J. Patel, and Alexander Serganov. "Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP." Proceedings of the National Academy of Sciences 112, no. 39 (September 15, 2015): E5391—E5400. http://dx.doi.org/10.1073/pnas.1515737112.

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Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-Å crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K+-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex–quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation–π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex–quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG–RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.

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Bieniasz, Paul, and Alice Telesnitsky. "Multiple, Switchable Protein:RNA Interactions Regulate Human Immunodeficiency Virus Type 1 Assembly." Annual Review of Virology 5, no. 1 (September 29, 2018): 165–83. http://dx.doi.org/10.1146/annurev-virology-092917-043448.

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Human immunodeficiency virus type 1 (HIV-1) particle assembly requires several protein:RNA interactions that vary widely in their character, from specific recognition of highly conserved and structured viral RNA elements to less specific interactions with variable RNA sequences. Genetic, biochemical, biophysical, and structural studies have illuminated how virion morphogenesis is accompanied by dramatic changes in the interactions among the protein and RNA virion components. The 5′ leader RNA element drives RNA recognition by Gag upon initiation of HIV-1 assembly and can assume variable conformations that influence translation, dimerization, and Gag recognition. As Gag multimerizes on the plasma membrane, forming immature particles, its RNA binding specificity transiently changes, enabling recognition of the A-rich composition of the viral genome. Initiation of assembly may also be regulated by occlusion of the membrane binding surface of Gag by tRNA. Finally, recent work has suggested that RNA interactions with viral enzymes may activate and ensure the accuracy of virion maturation.

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Pedrotti, Simona, Roberta Busà, Claudia Compagnucci, and Claudio Sette. "The RNA recognition motif protein RBM11 is a novel tissue-specific splicing regulator." Nucleic Acids Research 40, no. 3 (October 7, 2011): 1021–32. http://dx.doi.org/10.1093/nar/gkr819.

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Yu, Qingfen, Wei Ye, Cheng Jiang, Ray Luo, and Hai-Feng Chen. "Specific Recognition Mechanism between RNA and the KH3 Domain of Nova-2 Protein." Journal of Physical Chemistry B 118, no. 43 (October 21, 2014): 12426–34. http://dx.doi.org/10.1021/jp5079289.

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Ban, Ting, Jian-Kang Zhu, Karsten Melcher, and H. Eric Xu. "Structural mechanisms of RNA recognition: sequence-specific and non-specific RNA-binding proteins and the Cas9-RNA-DNA complex." Cellular and Molecular Life Sciences 72, no. 6 (November 29, 2014): 1045–58. http://dx.doi.org/10.1007/s00018-014-1779-9.

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Hu, Jianming, and Morgan Boyer. "Hepatitis B Virus Reverse Transcriptase and ε RNA Sequences Required for Specific Interaction In Vitro." Journal of Virology 80, no. 5 (March 1, 2006): 2141–50. http://dx.doi.org/10.1128/jvi.80.5.2141-2150.2006.

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ABSTRACT Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ε) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific ε binding, we have defined the protein and RNA sequences required for specific HBV RT-ε interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for ε binding. With respect to the ε RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of ε, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-ε interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-ε interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some ε sequences by the RT may be required specifically for viral DNA synthesis.

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Borkar, Aditi N., Michael F. Bardaro, Carlo Camilloni, Francesco A. Aprile, Gabriele Varani, and Michele Vendruscolo. "Structure of a low-population binding intermediate in protein-RNA recognition." Proceedings of the National Academy of Sciences 113, no. 26 (June 10, 2016): 7171–76. http://dx.doi.org/10.1073/pnas.1521349113.

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The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.

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Korn, Sophie M., Julian Von Ehr, Karthikeyan Dhamotharan, Jan-Niklas Tants, Rupert Abele, and Andreas Schlundt. "Insight into the Structural Basis for Dual Nucleic Acid—Recognition by the Scaffold Attachment Factor B2 Protein." International Journal of Molecular Sciences 24, no. 4 (February 7, 2023): 3286. http://dx.doi.org/10.3390/ijms24043286.

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The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately 100 kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA and RNA binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerize, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

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Schultz, Annemarie, Stephanie Nottrott, Nicholas James Watkins, and Reinhard Lührmann. "Protein-Protein and Protein-RNA Contacts both Contribute to the 15.5K-Mediated Assembly of the U4/U6 snRNP and the Box C/D snoRNPs." Molecular and Cellular Biology 26, no. 13 (July 1, 2006): 5146–54. http://dx.doi.org/10.1128/mcb.02374-05.

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ABSTRACT The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.

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Hake, Laura E., Raul Mendez, and Joel D. Richter. "Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger." Molecular and Cellular Biology 18, no. 2 (February 1, 1998): 685–93. http://dx.doi.org/10.1128/mcb.18.2.685.

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ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

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Yan, C., and T. Mélèse. "Multiple regions of NSR1 are sufficient for accumulation of a fusion protein within the nucleolus." Journal of Cell Biology 123, no. 5 (December 1, 1993): 1081–91. http://dx.doi.org/10.1083/jcb.123.5.1081.

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NSR1, a 67-kD nucleolar protein, was originally identified in our laboratory as a nuclear localization signal binding protein, and has subsequently been found to be involved in ribosome biogenesis. NSR1 has three regions: an acidic/serine-rich NH2 terminus, two RNA recognition motifs, and a glycine/arginine-rich COOH terminus. In this study we show that NSR1 itself has a bipartite nuclear localization sequence. Deletion of either basic amino acid stretch results in the mislocation of NSR1 to the cytoplasm. We further demonstrate that either of two regions, the NH2 terminus or both RNA recognition motifs, are sufficient to localize a bacterial protein, beta-galactosidase, to the nucleolus. Intensive deletion analysis has further defined a specific acidic/serine-rich region within the NH2 terminus as necessary for nucleolar accumulation rather than nucleolar targeting. In addition, deletion of either RNA recognition motif or point mutations in one of the RNP consensus octamers results in the mislocalization of a fusion protein within the nucleus. Although the glycine/arginine-rich region in the COOH terminus is not sufficient to bring beta-galactosidase to the nucleolus, our studies show that this domain is necessary for nucleolar accumulation when an RNP consensus octamer in one of the RNA recognition motifs is mutated. Our findings are consistent with the notion that nucleolar localization is a result of the binding interactions of various domains of NSR1 within the nucleolus rather than the presence of a specific nucleolar targeting signal.

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Roca-Martínez, Joel, Hrishikesh Dhondge, Michael Sattler, and Wim F. Vranken. "Deciphering the RRM-RNA recognition code: A computational analysis." PLOS Computational Biology 19, no. 1 (January 23, 2023): e1010859. http://dx.doi.org/10.1371/journal.pcbi.1010859.

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RNA recognition motifs (RRM) are the most prevalent class of RNA binding domains in eukaryotes. Their RNA binding preferences have been investigated for almost two decades, and even though some RRM domains are now very well described, their RNA recognition code has remained elusive. An increasing number of experimental structures of RRM-RNA complexes has become available in recent years. Here, we perform an in-depth computational analysis to derive an RNA recognition code for canonical RRMs. We present and validate a computational scoring method to estimate the binding between an RRM and a single stranded RNA, based on structural data from a carefully curated multiple sequence alignment, which can predict RRM binding RNA sequence motifs based on the RRM protein sequence. Given the importance and prevalence of RRMs in humans and other species, this tool could help design RNA binding motifs with uses in medical or synthetic biology applications, leading towards the de novo design of RRMs with specific RNA recognition.

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MacRae, Ian. "Structural basis for post-transcriptional gene silencing by human Argonaute-2." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1395. http://dx.doi.org/10.1107/s2053273314086045.

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Argonaute proteins are a unique class of RNases that degrade substrate RNAs in a sequence-specific manner. Argonaute proteins acquire substrate specificity by binding to a small RNA (21 nucleotide), termed the guide RNA, and use the encoded sequence to locate target message RNAs (mRNAs) through base-pairing complementarity. We have determined crystal structures of human Argonaute-2 (Ago2) bound a guide RNA and a variety of complementary target RNAs. The structures reveal how Ago2 uses discrete regions of the guide to scan for targets and the conformational changes associated with target recognition. Using free phenol as a probe, we also identified a constellation of hydrophobic cavities on the surface of Ago2 that we suggest are involved in the recruitment of additional protein factors to target mRNAs upon recognition by Ago2.

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33

Philippe, Bouvet, Ken Matsumoto, and Alan P. Wolffe. "Sequence-specific RNA Recognition by the Xenopus Y-box Proteins." Journal of Biological Chemistry 270, no. 47 (November 1995): 28297–303. http://dx.doi.org/10.1074/jbc.270.47.28297.

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Park, Sungmin, David G. Myszka, Michael Yu, Sarah J. Littler, and Ite A. Laird-Offringa. "HuD RNA Recognition Motifs Play Distinct Roles in the Formation of a Stable Complex with AU-Rich RNA." Molecular and Cellular Biology 20, no. 13 (July 1, 2000): 4765–72. http://dx.doi.org/10.1128/mcb.20.13.4765-4772.2000.

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ABSTRACT Human neuron-specific RNA-binding protein HuD belongs to the family of Hu proteins and consists of two N-terminal RNA recognition motifs (RRM1 and -2), a hinge region, and a C-terminal RRM (RRM3). Hu proteins can bind to AU-rich elements in the 3′ untranslated regions of unstable mRNAs, causing the stabilization of certain transcripts. We have studied the interaction between HuD and prototype mRNA instability elements of the sequence UU(AUUU)nAUU using equilibrium methods and real-time kinetics (surface plasmon resonance using a BIACORE). We show that a single molecule of HuD requires at least three AUUU repeats to bind tightly to the RNA. Deletion of RRM1 reduced theKd by 2 orders of magnitude and caused a decrease in the association rate and a strong increase in the dissociation rate of the RNA-protein complex, as expected when a critical RNA-binding domain is removed. In contrast, deletion of either RRM2 or -3, which only moderately reduced the affinity, caused marked increases in the association and dissociation rates. The slower binding and stabilization of the complex observed in the presence of all three RRMs suggest that a change in the tertiary structure occurs during binding. The individual RRMs bind poorly to the RNA (RRM1 binds with micromolar affinity, while the affinities of RRM2 and -3 are in the millimolar range). However, the combination of RRM1 and either RRM2 or RRM3 in the context of the protein allows binding with a nanomolar affinity. Thus, the three RRMs appear to cooperate not only to increase the affinity of the interaction but also to stabilize the formed complex. Kinetic effects, similar to those described here, could play a role in RNA binding by many multi-RRM proteins and may influence the competition between proteins for RNA-binding sites and the ability of RNA-bound proteins to be transported intracellularly.

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Saurer, Martin, Marc Leibundgut, Hima Priyanka Nadimpalli, Alain Scaiola, Tanja Schönhut, Richard G. Lee, Stefan J. Siira, et al. "Molecular basis of translation termination at noncanonical stop codons in human mitochondria." Science 380, no. 6644 (May 5, 2023): 531–36. http://dx.doi.org/10.1126/science.adf9890.

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The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo–electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

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36

Lindsey-Boltz, L. A., and A. Sancar. "RNA polymerase: The most specific damage recognition protein in cellular responses to DNA damage?" Proceedings of the National Academy of Sciences 104, no. 33 (August 7, 2007): 13213–14. http://dx.doi.org/10.1073/pnas.0706316104.

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37

Nevinsky, Georgy A. "How Enzymes, Proteins, and Antibodies Recognize Extended DNAs; General Regularities." International Journal of Molecular Sciences 22, no. 3 (January 29, 2021): 1369. http://dx.doi.org/10.3390/ijms22031369.

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X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their total affinity for enzymes and proteins. The interaction of different enzymes and proteins with long DNA and RNA at the quantitative molecular level can be successfully analyzed using the method of the stepwise increase in ligand complexity (SILC). The present review summarizes the data on stepwise increase in ligand complexity (SILC) analysis of nucleic acid recognition by various enzymes—replication, restriction, integration, topoisomerization, six different repair enzymes (uracil DNA glycosylase, Fpg protein from Escherichia coli, human 8-oxoguanine-DNA glycosylase, human apurinic/apyrimidinic endonuclease, RecA protein, and DNA-ligase), and five DNA-recognizing proteins (RNA helicase, human lactoferrin, alfa-lactalbumin, human blood albumin, and IgGs against DNA). The relative contributions of structural elements of DNA fragments “covered” by globules of enzymes and proteins to the total affinity of DNA have been evaluated. Thermodynamic and catalytic factors providing discrimination of unspecific and specific DNAs by these enzymes on the stages of primary complex formation following changes in enzymes and DNAs or RNAs conformations and direct processing of the catalysis of the reactions were found. General regularities of recognition of nucleic acid by DNA-dependent enzymes, proteins, and antibodies were established.

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38

Venter, P. Arno, and Anette Schneemann. "Assembly of Two Independent Populations of Flock House Virus Particles with Distinct RNA Packaging Characteristics in the Same Cell." Journal of Virology 81, no. 2 (November 1, 2006): 613–19. http://dx.doi.org/10.1128/jvi.01668-06.

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ABSTRACT Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.

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39

Freisz, Séverine, Joelle Mezher, Lamine Hafirassou, Philippe Wolff, Yves Nominé, Christophe Romier, Philippe Dumas, and Eric Ennifar. "Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein." RNA Biology 9, no. 7 (July 2012): 966–77. http://dx.doi.org/10.4161/rna.20483.

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40

Ptacek, Jakub, Dong Zhang, Liming Qiu, Sven Kruspe, Lucia Motlova, Petr Kolenko, Zora Novakova, et al. "Structural basis of prostate-specific membrane antigen recognition by the A9g RNA aptamer." Nucleic Acids Research 48, no. 19 (June 11, 2020): 11130–45. http://dx.doi.org/10.1093/nar/gkaa494.

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Abstract Prostate-specific membrane antigen (PSMA) is a well-characterized tumor marker associated with prostate cancer and neovasculature of most solid tumors. PSMA-specific ligands are thus being developed to deliver imaging or therapeutic agents to cancer cells. Here, we report on a crystal structure of human PSMA in complex with A9g, a 43-bp PSMA-specific RNA aptamer, that was determined to the 2.2 Å resolution limit. The analysis of the PSMA/aptamer interface allows for identification of key interactions critical for nanomolar binding affinity and high selectivity of A9g for human PSMA. Combined with in silico modeling, site-directed mutagenesis, inhibition experiments and cell-based assays, the structure also provides an insight into structural changes of the aptamer and PSMA upon complex formation, mechanistic explanation for inhibition of the PSMA enzymatic activity by A9g as well as its ligand-selective competition with small molecules targeting the internal pocket of the enzyme. Additionally, comparison with published protein–RNA aptamer structures pointed toward more general features governing protein-aptamer interactions. Finally, our findings can be exploited for the structure-assisted design of future A9g-based derivatives with improved binding and stability characteristics.

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41

Grotwinkel, Jan Timo, Klemens Wild, Bernd Segnitz, and Irmgard Sinning. "SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation." Science 344, no. 6179 (April 3, 2014): 101–4. http://dx.doi.org/10.1126/science.1249094.

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The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

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42

Kazlovskiy, I. S., and M. A. Zinchenko. "Construction of a strain-producer of the chimeric protein consisting of RNA polymerase and a DNA-affinity domain." Doklady of the National Academy of Sciences of Belarus 62, no. 5 (October 30, 2018): 601–7. http://dx.doi.org/10.29235/1561-8323-2018-62-5-601-607.

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One of the recent perspective trends of molecular biotechnology is cell-free synthesis of protein. The procedure of cell-free synthesis of protein is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes. Procreation of the transcription stage requires participation of specific RNA polymerase which initiates process of mRNA synthesis from the particular sites of recognition. Often the DNA-dependent RNA polymerase of a bacteriophage of T7 (T7 RNA polymerase) is for this purpose applied. For improvement of qualitative characteristics of the T7 RNA polymerase in the real work the new strain of Escherichia coli producing this enzyme fused with the DNA-affine Sso7d domain of a thermophilic bacterium Sulfolobus solfataricus is created. The producing ability of the received recombinant strain concerning synthesized chimera protein reaches 625 un/l of cultural liquid, and the specific activity of the purified enzyme preparation was 80 un/ μg of protein. The received enzyme is intended for use as tools at synthesis of proteins in cell-free system.

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43

Levine, T. D., F. Gao, P. H. King, L. G. Andrews, and J. D. Keene. "Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs." Molecular and Cellular Biology 13, no. 6 (June 1993): 3494–504. http://dx.doi.org/10.1128/mcb.13.6.3494-3504.1993.

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We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

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Levine, T. D., F. Gao, P. H. King, L. G. Andrews, and J. D. Keene. "Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs." Molecular and Cellular Biology 13, no. 6 (June 1993): 3494–504. http://dx.doi.org/10.1128/mcb.13.6.3494.

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We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

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45

Bolla, Jani Reddy, Anna C. Howes, Francesco Fiorentino, and Carol V. Robinson. "Assembly and regulation of the chlorhexidine-specific efflux pump AceI." Proceedings of the National Academy of Sciences 117, no. 29 (July 7, 2020): 17011–18. http://dx.doi.org/10.1073/pnas.2003271117.

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Few antibiotics are effective againstAcinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combatA. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacterchlorhexidine efflux protein I), is encoded for by theaceIgene and is under the transcriptional control of AceR (Acinetobacterchlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.

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Kitamura, Sayaka, Kosuke Fujishima, Asako Sato, Daisuke Tsuchiya, Masaru Tomita, and Akio Kanai. "Characterization of RNase HII substrate recognition using RNase HII–argonaute chimaeric enzymes from Pyrococcus furiosus." Biochemical Journal 426, no. 3 (February 24, 2010): 337–44. http://dx.doi.org/10.1042/bj20091553.

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RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA–DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA–DNA hybrids than for RNA–RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA–RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth β-sheet and the fifth α-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII–Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

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Yu, Michael C. "The Role of Protein Arginine Methylation in mRNP Dynamics." Molecular Biology International 2011 (April 7, 2011): 1–10. http://dx.doi.org/10.4061/2011/163827.

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In eukaryotes, messenger RNA biogenesis depends on the ordered and precise assembly of a nuclear messenger ribonucleoprotein particle (mRNP) during transcription. This process requires a well-orchestrated and dynamic sequence of molecular recognition events by specific RNA-binding proteins. Arginine methylation is a posttranslational modification found in a plethora of RNA-binding proteins responsible for mRNP biogenesis. These RNA-binding proteins include both heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) proteins. In this paper, I discuss the mechanisms of action by which arginine methylation modulates various facets of mRNP biogenesis, and how the collective consequences of this modification impart the specificity required to generate a mature, translational- and export-competent mRNP.

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48

Hillen, Hauke S., Dmitriy A. Markov, Ireneusz D. Wojtas, Katharina B. Hofmann, Michael Lidschreiber, Andrew T. Cowan, Julia L. Jones, Dmitry Temiakov, Patrick Cramer, and Michael Anikin. "The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3′-UTRs of yeast mitochondrial mRNAs." Proceedings of the National Academy of Sciences 118, no. 15 (April 5, 2021): e2009329118. http://dx.doi.org/10.1073/pnas.2009329118.

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Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5′ capping and 3′ polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3′-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3′-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.

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Eichhorn, Catherine D., Yuan Yang, Lucas Repeta, and Juli Feigon. "Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7." Proceedings of the National Academy of Sciences 115, no. 28 (June 26, 2018): E6457—E6466. http://dx.doi.org/10.1073/pnas.1806276115.

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The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein–RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3′OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13C spin relaxation data and comparison of free xRRM, RNA, and xRRM–RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM–7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3′ end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

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Schaal, Thomas D., and Tom Maniatis. "Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1705–19. http://dx.doi.org/10.1128/mcb.19.3.1705.

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ABSTRACT Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

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Journal articles on the topic 'Specific protein/RNA recognition'

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