Littérature scientifique sur le sujet « Ribonucleoprotein particles RNPs »

Ro ribonucleoprotein particles (Ro RNPs) are complexes of several proteins with a small RNA polymerase III-transcribed Ro RNA. Despite their relative abundance and evolutionary conservation no function has as yet been ascribed to these complexes. Also their subcellular distribution is still largely unknown as immunofluorescence studies concerning their localization have produced conflicting data. We have used cell enucleation to fractionate cells into cytoplasmic and nuclear fractions. Analysis of these fractions revealed an exclusively cytoplasmic localization for the Ro RNPs. The majority of the Ro RNAs are shown to be stably associated with all three known Ro RNP proteins. Although no Ro RNAs could be detected in the nuclear fraction, the Ro RNP-specific proteins were abundantly present. These nuclear non-Ro RNA-associated proteins are shown to be capable of binding Ro RNAs.

AbstractSmall nucleolar and Cajal body ribonucleoprotein particles (RNPs) are required for the maturation of ribosomes and spliceosomes. They consist of small nucleolar RNA or Cajal body RNA combined with partner proteins and represent the most complex RNA modification enzymes. Recent advances in structure and function studies have revealed detailed information regarding ribonucleoprotein assembly and substrate binding. These enzymes form intertwined RNA–protein assemblies that facilitate reversible binding of the large ribosomal RNA or small nuclear RNA. These revelations explain the specificity among the components in enzyme assembly and substrate modification. The multiple conformations of individual components and those of complete RNPs suggest a dynamic assembly process and justify the requirement of many assembly factors in vivo.

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.

Abstract Ro ribonucleoprotein particles (Ro RNPs) are evolutionarily conserved cytoplasmic complexes of unknown function. They are composed of several proteins and a small, RNA polymerase III-transcribed Ro or Y RNA. Abs directed against the protein moiety of Ro RNPs are often found in sera of patients suffering from certain autoimmune disorders. The association of one of the Ro proteins, a protein of 52 kDa (Ro52), with Ro RNPs is still questionable. In this study, we have used anti-Ro52 Abs isolated from autoimmune sera to locate the antigenic determinants of Ro52 and to analyze the correlation between regions of Ro52 recognized by these Abs and their ability to immunoprecipitate Ro RNPs. The results indicate that the autoimmune response against Ro52 is heterogeneous and that the exclusive recognition of certain epitopes does not result in immunoprecipitation of Ro RNPs.

Proteins and RNA are often found in ribonucleoprotein particles (RNPs), where they function in cellular processes to synthesize proteins (the ribosome), chemically modify RNAs (small nucleolar RNPs), splice pre-mRNAs (the spliceosome), and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli). Each RNA–protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.

45 S RNP (ribonucleoprotein) particles from calf thymus or L5178y mouse lymphoma cells contain the poly(A)-modulated and oligo(U)-binding endoribonuclease VII [Bachmann, Zahn & Müller (1983) J. Biol. Chem. 258, 7033-7040]. From these particles a 4.5 S RNA was isolated that possesses an oligo(U) sequence. By using monospecific and non-cross-reacting antibodies directed against the La or Ro antigen, both proteins were identified in the endoribonuclease VII-RNP complex after phosphorylation in vitro. In a second approach, endoribonuclease VII activity was identified in immunoaffinity-purified Ro RNPs after preparative isoelectric focusing. Therefore we conclude that the 4.5 S RNA belongs to the Ro RNAs. The results indicate a possible function of endoribonuclease VII in activating stored mRNAs.

Balbiani ring (BR) granules are premessenger ribonucleoprotein particles (RNPs) generated in giant chromosomal puffs, the BRs, in the larval salivary glands of the dipteran chironomus tentans. Monoclonal antibodies were raised against nuclear proteins collected on a single-stranded-DNA-agarose affinity column, and two of them were used to identify RNA-binding proteins in BR granules. First, in Western blots (immunoblots), one of the antibodies recognized a 36-kDa protein and the other recognized a 45-KDa protein. Second, both antibodies bound to the BRs in immunocytological experiments. It was shown in cross-linking experiments that the two proteins are associated with heterogeneous nuclear RNP (hnRNP) complexes extracted from C. tentans nuclei. By immunoelectron microscopy of isolated and partly unfolded BR RNPs, it was specifically demonstrated that the BR granules contain the two proteins and, in addition, that both proteins are distributed frequently along the RNP fiber of the particles. Thus, the 36- and 45-KDa proteins are likely to be abundant, RNA-binding proteins in the BR particles. To elucidate to what extent the two proteins are also present in other hnRNPs, we studied the binding of the antibodies to chromosomal puffs in general. It was observed that many puffs in addition to the BRs harbor the two proteins, but there are also puffs containing only one of the components, either the 36- or the 45-kDa protein. We conclude that the two proteins are not randomly bound to all hnRNPs but that each of them seems to be linked to a specific subset of the particles.

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.

We first reported on the existence of vault ribonucleoprotein particles in 1986, and since this study we have demonstrated that these unusual RNPs are ubiquitously expressed and highly conserved among diverse eukaryotes. These particles are quite large (65 x 35 nm) and distinctly regular in shape and dimensions. The polypeptide composition of vaults is also similar between species, dominated by a∼100 Kd protein which makes up >70% of the particles mass. The RNA component of vaults, which has been sequenced and characterized from both rat and bullfrog, does not appear to serve a structural role, and due to the strong conservation of its secondary structure could serve a functional role.

Ubiquitination is one of the most abundant and versatile post-translation modifications in eukaryotes that affects many biological processes by modifying protein activity, interactions, localization and stability of substrates. E3 ligases have a key function in the process, acting as molecular ubiquitin-substrate matchmakers and providing specificity to the reaction. In this thesis, we aimed at characterizing the physiological and pathological functions of the human HECW1, a poorly studied E3 ligase which belongs to the NEDD4 family. HECW1 is preferentially expressed in the central nervous system (CNS) and it has been linked to neurodegeneration, in particular to the familial form of Amyotrophic Lateral Sclerosis (fALS). The Drosophila orthologue Hecw, that we recently identified and functionally characterized, is similarly enriched in the CNS and is involved in the dynamic regulation of RNPs required for neuronal health. The Hecw/HECW1 interactome is enriched in proteins involved in the autophagy/endolysosomal pathway and RNPs dynamics, whose dysfunction promote neurodegenerative diseases. Together these data suggest a protective role of HECW1 in neuronal homeostasis. To investigate into HECW1 physiological and disease-relevant neuronal function, we optimized a protocol to directly differentiate neurons from human iPSCs and we generated HECW1-KO iPSCs. We found that HECW1 expression in neurons is upregulated during differentiation and downregulated with aging, a typical behavior of components of the ubiquitin proteasome and autophagy pathways. Unbiased proteomic analysis showed deregulation of proteins involved in vesicle traffic and kinase activity in HECW-KO neurons. Targeted immunofluorescence, morphological and EM analysis revealed an accumulation of enlarged organelles positive for the autophagic and endo-lysosomal marker LAMP1 and of endolysosomal/autophagic compartments structures along filaments and in distal axons of HECW1-depleted neurons. Moreover, distal tips of HECW1-KO neurons showed the accumulation of abnormal, static WGA-aggregates, indicating an impairment in endosomal traffic. A second category of enriched HECW1 interactors is related to RNA metabolism. Co-immunoprecipitation analysis confirmed HECW1 interaction with the SG protein FMRP and the PB component EDC3. We also measured an increase number of constitutive PBs in HECW1-depleted neurons, suggesting a possible regulatory role of HECW1 in constitutive PB formation or clearance. The two phenotypes observed in HECW1-depleted neurons could be functionally linked, considering the involvement of autophagy in the clearance of persistent RNPs arising from chronic stress or disease mutations. Taken together, our results have uncovered an involvement of HECW1 in the regulation of the autophagy/endolysosomal pathway in neurons and a possible contribution in controlling the homeostasis of ribonucleoprotein particles. Future studies in motor neurons would help to understand the implications of these results for ASL, a disease where dysregulation of RNA metabolism, cytoplasmic mislocalization of RNA binding proteins and dysfunction in RNP dynamics, along with autophagy impairments appear to be at the basis of the pathogenesis.

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.

Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levuriennes Dim1 et Bud23-Trm112, qu’elles sont requises pour la synthèse et la modification de l’ARNr mature de la petite sous-unité ribosomique, et qu’elles seraient impliquées dans un mécanisme conservé contrôlant la qualité de la voie de biosynthèse du ribosome.

La totalité des FA que nous avons identifiés en cellule humaine sont à la disposition de la communauté scientifique dans une base de données en ligne accessible sur la page www.RibosomeSynthesis.com. Nous espérons que cette ressource contribuera à une meilleure compréhension des mécanismes moléculaires sous-jacents au développement des ribosomopathies et à l’élaboration d’agents thérapeutiques efficaces.

Doctorat en sciences, Spécialisation biologie moléculaire
info:eu-repo/semantics/nonPublished

Most protein-encoding genes in Eukaryotes are separated into alternating coding and non-coding sequences (exons and introns). Following the transcription of the DNA into pre-messenger RNA (pre-mRNA) in the nucleus, a macromolecular complex termed spliceosome removes the introns and joins the exons to generate mature mRNA that is exported to the cytoplasm. There, it can be interpreted by ribosomes to generate proteins. The spliceosome consists of five small nuclear ribonucleic acids (snRNAs) and more than 150 proteins. Integral components of this complex are RNA-protein particles (RNPs) composed of one or two snRNAs, seven common (Sm) and a various number of snRNP-specific proteins. The Sm proteins form a ring-structure around a conserved site of the snRNA called Sm site. In vitro, Sm proteins (B/B', D1, D2, D3, E, F, G) and snRNA readily assemble to form snRNPs. In the context of the cell, however, two macromolecular trans-acting factors, the PRMT5 (protein arginine methyltransferases type 5) and the SMN (survival motor neuron) complex, are needed to enable this process. Initially, the Sm proteins in the form of heterooligomers D1/D2, D3/B and F/E/G are sequestered by the type II methyltransferase PRMT5. pICln, a component of the PRMT5 complex, readily interacts with Sm proteins to form two distinct complexes. Whereas the first one comprises pICln and D3/B the second one forms a ring consisting of pICln, D1/D2 and F/E/G (6S). It has been found that pICln prevents the premature interaction of snRNAs with the Sm proteins in these complexes and thus functions as an assembly chaperone imposing a kinetic trap upon the further assembly of snRNPs. PRMT5 catalyzes the symmetrical dimethylation of arginine residues in B/B', D1 and D3 increasing their affinity towards the SMN complex. Finally, the SMN complex interacts with the pICln-Sm protein complexes, expels pICln and mediates snRNP assembly in an ATP-dependent reaction. So far, only little is known about the action of PRMT5 in the early phase of snRNP assembly and especially how the 6S complex is formed. Studies of this have so far been hampered by the unavailability of soluble and biologically active PRMT5 enzyme. The composition of the SMN complex and possible functions of individual subunits have been elucidated or hypothesized in recent years. Still, the exact mechanism of the entire machinery forming snRNPs is poorly understood. In vivo, reduced production of functional SMN protein results in the neurodegenerative disease spinal muscular atrophy (SMA). How specific SMN mutations that have been found in SMA patients cause the disease remains elusive, yet, are likely to interfere with either SMN complex stability or snRNP assembly. The aim of this work was to establish an in vitro system to recapitulate the cytoplasmic assembly of snRNPs. This was enabled by the recombinant production of all PRMT5 and SMN complex components as well as Sm proteins in a combination of bacterial and insect cell expression systems. Co-expression of human PRMT5 and its direct interaction partner WD45 (WD-repeat domain 45) in Sf21 (Spodoptera frugiperda 21) insect cells resulted for the first time in soluble and biologically active enzyme. Recombinant PRMT5/WD45 formed complexes with Sm protein heterooligomers as well as pICln-Sm protein complexes but not with F/E/G alone. Also, the enzyme exhibited a type II methyltransferase activity catalyzing the mono- (MMA) and symmetrical dimethylation (sDMA) of Sm proteins B, D1 and D3. Two experimental setups were devised to quantitatively analyze the overall methylation of substrates as well as to identify the type and relative abundance of specific methylation types. Methylation of Sm proteins followed Michaelis-Menten kinetics. Complex reconstitutions and competition of the methylation reaction indicate that 6S is formed in a step-wise manner on the PRMT5 complex. The analysis of the methylation type could be applied to deduce a model of sequential MMA and sDMA formation. It was found that large Sm protein substrate concentrations favored monomethylation. Following a distributive mechanism this leads to the conclusion that PRMT5 most likely confers partial methylation of several different substrate proteins instead of processing a single substrate iteratively until it is completely dimethylated. Finally, the human SMN complex was reconstituted from recombinant sources and was shown to be active in snRNP formation. The introduction of a modified SMN protein carrying a mutation (E134K) present in spinal muscular atrophy (SMA) proved that mutated complexes can be generated in vitro and that these might be applied to elucidate the molecular etiology of this devastating disease
Der Großteil der Protein-kodierenden Gene in Eukaryoten ist in kodierende und nicht-kodierende Regionen unterteilt - sogenannte Exons und Introns. Damit aus einem Gen ein Protein hergestellt werden kann, muss zunächst die genomische DNA im Rahmen der Translation in prä-messenger RNA (prä-mRNA; Boten-RNA) übersetzt werden. Aus dieser prä-mRNA werden anschließend durch einen makromolekularen Komplex (Spleißosom) die Introns entfernt und die kodieren Exons zusammengefügt. Die daraus resultierende gereifte mRNA dient letztendlich den Ribosomen als Vorlage zur Herstellung von Proteinen. Das Spleißosom besteht aus fünf snRNAs (small nuclear ribonucleic acids) und über 150 weiteren Proteinen. Zentrale Komponenten dieses Komplexes sind RNA-Protein Partikel (RNPs), die aus einer bzw. zwei snRNAs, sieben gemeinsamen (Sm) und weiteren snRNP-spezifischen Proteinen bestehen. Die Sm Proteine (B/B', D1, D2, D3, E, F and G) bilden eine Ringstruktur um eine konservierte Sequenz (Sm-site) der snRNA aus. In vitro erfolgt die Ausbildung dieser Struktur spontan. Im zellulären Kontext wird die Zusammenlagerung dieser snRNPs allerdings erst durch zwei makromolekulare, trans-agierende Proteinkomplexe, den PRMT5 und den SMN Komplex, ermöglicht. Zu Beginn interagieren die Sm Proteine als heterooligomere Strukturen bestehend aus D1/D2, D3/B und F/E/G mit der Typ II Methyltransferase PRMT5. pICln, eine Komponente des PRMT5 Komplexes, interagiert mit den Sm Proteinen und bildet zwei spezifische Komplexe aus. Während der erste aus pICln und D3/B besteht, lagern sich im zweiten die Sm proteine D1/D2 und F/E/G mit pICln zu einem Ring zusammen (6S Komplex). Diese Interaktion erzeugt eine kinetische Falle, so dass die Sm Proteine sich nicht mehr spontan an die snRNA anlagern können und somit die snRNP Biogenese verzögert wird. PRMT5 katalysiert die symmetrische Dimethylierung von Argininresten in B/B', D1 und D3, wodurch deren Affinität zum SMN Komplex erhöht wird. Letztendlich assoziert der SMN Komplex mit den zuvor erzeugten pICln-Sm Protein Komplexen, entlässt pICln und ermöglicht im weiteren die Zusammenlagerung von snRNPs in einer ATP-abhängigen Reaktion. Aktuell ist über die Funktion von PRMT5 in der frühen Phase der snRNP Biogenese wenig bekannt. Dies trifft insbesondere auf die Zusammenlagerung des 6S Komplexes zu. Biochemische Untersuchungen waren bis jetzt nahezu unmöglich, da rekombinant hergestelltes Protein entweder unlöslich oder biochemisch inaktiv war. In den vergangenen Jahren wurde viel über die Zusammensetzung des SMN Komplexes sowie über die Funktionen einzelner Untereinheiten herausgefunden aber auch spekuliert. Trotz alledem ist der genaue Mechanismus der snRNP Biogenese noch nahezu unbekannt. In vivo sind verringerte Mengen an funktionalem SMN Protein der Ausschlaggeber für die neurodegenerative Krankheit Spinale Muskelatrophie (SMA). Welchen Effekt Mutationen im SMN Protein haben, die in SMA Patienten festgestellt wurden ist ungewiss. Es ist allerdings zu vermuten, dass diese entweder die Integrität des SMN Komplexes negativ beeinflussen oder störend auf die snRNP Biogenese wirken. Das Ziel dieser Arbeit war es ein in vitro-System zu generieren, um die zytoplasmatische snRNP Biogenese biochemisch zu untersuchen. Dies geschah durch die rekombinante Produktion aller PRMT5 und SMN Komplex Komponenten sowie der Sm Proteine in einer Kombination von bakterieller und Insektenzell-Expression. Durch die Ko-Expression von humanem PRMT5 und dem Interaktionspartner WD45 (WD-repeat domain 45) in Sf21 (Spodoptera frugiperda 21) Insekten Zellen konnte erstmals lösliches und enzymatisch aktives Protein hergestellt werden. Rekombinantes PRMT5/WD45 bildete Komplexe mit heterooligomeren Sm Proteinen sowie pICln-Sm Protein Komplexen, allerdings nicht mit F/E/G. Zusätzlich konnte eine Typ II Methyltransferase Aktivität dadurch nachgewiesen werden, dass die Sm Protein B, D1 und D3 monomethyliert (MMA) und symmetrisch dimethyliert (sDMA) werden können. Zur weiteren Untersuchung wurden zwei experimentelle Ansätze erarbeitet, um die allgemeine Methylierungsaktivität sowie das relative Vorhandensein von Mono- und Dimethylargininen zu bestimmen. Es konnte gezeigt werden, dass die Methylierung der Sm Proteine einer Michael-Menten Kinetik folgt. Die Rekonstitution von PRMT-Sm Protein Komplexen sowie the Methylierungsreaktionen deuten auf eine schrittweise Zusammenlagerung von 6S auf dem PRMT5 Komplex hin.

AbstractMicrococcal nuclease (MNase) originating from Staphylococcus aureus is a calcium dependent ribo- and desoxyribonuclease which has endo- and exonucleolytic activity of low sequence preference. MNase is widely used to analyze nucleosome positions in chromatin by probing the enzyme’s DNA accessibility in limited digestion reactions. Probing reactions can be performed in a global way by addition of exogenous MNase, or locally by “chromatin endogenous cleavage” (ChEC) reactions using MNasefusion proteins. The latter approach has recently been adopted for the analysis of local RNA environments of MNasefusion proteins which are incorporated in vivo at specific sites of ribonucleoprotein (RNP) complexes. In this case, ex vivo activation of MNase by addition of calcium leads to RNA cleavages in proximity to the tethered anchor protein thus providing information about the folding state of its RNA environment.Here, we describe a set of plasmids that can be used as template for PCR-based MNase tagging of genes by homologous recombination in S. cerevisiae. The templates enable both N- and C-terminal tagging with MNase in combination with linker regions of different lengths and properties. In addition, an affinity tag is included in the recombination cassettes which can be used for purification of the particle of interest before or after induction of MNase cleavages in the surrounding RNA or DNA. A step-by-step protocol is provided for tagging of a gene of interest, followed by affinity purification of the resulting fusion protein together with associated RNA and subsequent induction of local MNase cleavages.

Metabolism of messenger RNA (mRNA) is critical for control of cell phenotype and includes several steps: transcription of DNA into a pre-mRNA, mRNA maturation, nucleocytoplasmic export and transport to specific cellular locations, translation into proteins, and decay. All these steps are seamlessly integrated and controlled by a large number of RNA-binding proteins that interact with RNA, forming messenger ribonucleoprotein particles. Several noncoding RNAs, such as microRNAs, also regulate mRNA metabolism. Activity-dependent control of mRNA transcription, splicing, and translation are critical for growth, plasticity, and repair in the nervous system. Disorders of RNA metabolism are a major disease pathway for a large number of neurologic disorders, many of them associated with accumulation of stress granules containing RNA and associated proteins. Elucidation of the pathophysiology of some of these disorders provides novel approaches for their treatment, including antisense oligonucleotide therapy.