Littérature scientifique sur le sujet « Intronic polyadenylation »

Most eukaryotic messenger RNAs are capped, spliced, and polyadenylated via co-transcriptional processes that are coupled to each other and to the transcription machinery. Coordination of these processes ensures correct RNA maturation and provides for the diversity of the transcribed isoforms. Thus, RNA processing is a chain of events in which the completion of one event is coupled to the initiation of the next one. In this context, the relationship between splicing and polyadenylation is an important aspect of gene regulation. We have found that cryptic polyadenylation signals are widely distributed over the intron sequences of Drosophila melanogaster. As shown by analyzing the distribution of genes arranged in a nested pattern, where one gene is fully located within an intron of another gene, overlapping of putative polyadenylation signals is a fairly common event affecting about 17% of all genes. Here we show that polyadenylation signals are silenced within introns: the poly(A) signal is utilized in the exonic but not in the intronic regions of the transcript. The transcription does not end within the introns, either in a transient reporter system or in the genomic context, while deletion of the 5'-splice site restores their functionality. According to a full Drosophila transcriptome analysis, utilization of intronic polyadenylation signals occurs very rarely and such events are likely to be inducible. These results confirm that the transcription apparatus ignores premature polyadenylation signals for as long as they are intronic.

Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3’ UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.

ABSTRACT Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.

Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.

Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.

In several eukaryotic organisms, heterochromatin (HC) in the introns of genes can regulate RNA processing, including polyadenylation, but the mechanism underlying this regulation is poorly understood. By promoting distal polyadenylation, the bromo-adjacent homology (BAH) domain-containing and RNA recognition motif-containing protein ASI1 and the H3K9me2-binding protein EDM2 are required for the expression of functional full-length transcripts of intronic HC-containing genes in Arabidopsis. Here we report that ASI1 and EDM2 form a protein complex in vivo via a bridge protein, ASI1-Immunoprecipitated Protein 1 (AIPP1), which is another RNA recognition motif-containing protein. The complex also may contain the Pol II CTD phosphatase CPL2, the plant homeodomain-containing protein AIPP2, and another BAH domain protein, AIPP3. As is the case with dysfunction of ASI1 and EDM2, dysfunction of AIPP1 impedes the use of distal polyadenylation sites at tested intronic HC-containing genes, such as the histone demethylase gene IBM1, resulting in a lack of functional full-length transcripts. A mutation in AIPP1 causes silencing of the 35S-SUC2 transgene and genome-wide CHG hypermethylation at gene body regions, consistent with the lack of full-length functional IBM1 transcripts in the mutant. Interestingly, compared with asi1, edm2, and aipp1 mutations, mutations in CPL2, AIPP2, and AIPP3 cause the opposite effects on the expression of intronic HC-containing genes and other genes, suggesting that CPL2, AIPP2, and AIPP3 may form a distinct subcomplex. These results advance our understanding of the interplay between heterochromatic epigenetic modifications and RNA processing in higher eukaryotes.

Abstract Summary Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. APA is dynamically regulated under different growth and differentiation conditions. Here, we present a bioinformatics package, named APAlyzer, for examining 3′UTR APA, intronic APA and gene expression changes using RNA-seq data and annotated polyadenylation sites in the PolyA_DB database. Using APAlyzer and data from the GTEx database, we present APA profiles across human tissues. Availability and implementation APAlyzer is freely available at https://bioconductor.org/packages/release/bioc/html/APAlyzer.html as an R/Bioconductor package. Supplementary information Supplementary data are available at Bioinformatics online.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. Vita.
Includes bibliographical references.
During transcription, cyclin-dependent kinases (CDKs) dynamically phosphorylate the C-terminal domain (CTD) of RNA Polymerase II (RNAPII) to recruit factors that coordinate transcription and mRNA biogenesis. Cdk12 phosphorylates Serine 2 (Ser2) of the RNAPII CTD, a modification associated with the regulation of transcription elongation, splicing, and cleavage/polyadenylation. Unlike other transcriptional CDKs that regulate most expressed genes, Cdk12 depletion abrogates the expression of homologous recombination (HR) genes relatively specifically, suppressing the HR DNA damage repair pathway and sensitizing cells to genotoxic stresses that cause replication fork collapse, such as Parp1 inhibitors. The proposed role for Cdk12 in regulating HR is clinically significant for two reasons. First, Cdk12 loss-of-function mutations populate high-grade serous ovarian carcinoma and castration-resistant prostate tumors raising the possibility that Cdk12 mutational status may predict the effectiveness of chemotherapeutics that target HR-deficient tumors. Second, readily available small molecule inhibitors of Cdk12 induce sensitization of HR-competent tumors to Parp1 inhibitors in vivo raising the possibility that inhibitors against Cdk12 could be used as chemotherapeutics. Despite this growing clinical interest, the mechanism behind Cdk12's regulation of HR genes remains unknown. Here we show that Cdk12 suppresses intronic polyadenylation (IPA) and that this mechanism explains the exquisite sensitivity of HR genes to Cdk12 loss. We find that Cdk12 globally enhances transcription elongation rate to kinetically suppress IPA events. Many HR genes harbor multiple IPA sites per gene, and the cumulative effect of these sites accounts for the increased sensitivity of HR genes to Cdk12. Finally, we find evidence that Cdk12 LOF mutations and deletions cause upregulation of IPA sites in HR genes in human tumors. Our results define the mechanism by which Cdk12 regulates transcription, mRNA biogenesis, and the HR pathway. This work clarifies the biological function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumor biomarker.
by Sara Jane Dubbury.
Ph. D.

Au cours d’études sur la polyadénylation alternative (APA), des transcrits courts terminant dans un exon final alternatif ont été découverts, on parle de polyadénylation intronique (IPA). L’IPA est régulée par des facteurs de l’épissage (dont U1 snRNP), de polyadénylation, et d’élongation de la transcription (dont CDK12). Les isoformes IPA sont régulées par des agents génotoxiques (induisant des dommages à l’ADN), dont les rayonnements UV et la doxorubicine. Les inhibiteurs de CDK12 augmentent l’IPA dans des gènes de réparation de l’ADN et la sensibilité cellulaire à des génotoxiques. L’IPA a souvent lieu dans la région codante des gènes, générant des protéines altérées en carboxy terminal. Cependant, des transcrits IPA sont aussi générés dans les premiers introns des gènes, on parle alors de 5’IPA. Des transcrits 5’IPA sont dégradés par l’exosome nucléaire, mais certains sont abondants et ont un faible potentiel codant. Deux d’entre eux, issues des gènes ASCC3 et CDKN1A, ont des fonctions non codantes. Par ailleurs, des études montrent par Ribo-seq et spectrométrie de masse (MS) l’existence, dans des ARNm et des lncRNA, de petits cadres de lecture ouverts (sORF) codant des microprotéines (miP, protéines de moins de 100 aa) qui peuvent être fonctionnelles. Aucune miP n’a été rapportée dans des isoformes 5’IPA. Le cisplatine (CisPt) est un agent pontant de l'ADN très utilisé dans les cancers du poumon non à petites cellules (NSCLC). Mon équipe a observé par 3’-seq dans des cellules NSCLC que le CisPt augmente l’expression des isoformes IPA par rapport aux ARNm canoniques (ratio IPA:LE) dans de nombreux gènes, et que certaines isoformes IPA sont peu engagées dans les polysomes lourds et sont issues de la région en amont du site d’initiation de la traduction annoté du gène (isoformes 5’UTR-IPA). Mes objectifs étaient de déterminer le rôle de l’IPA dans la réponse cellulaire au CisPt. Je me suis intéressé premièrement au rôle des isoformes 5’UTR-IPA. Pour deux d’entre elles, issues des gènes PRKAR1B et PHF20, j’ai montré que leur déplétion par siARN augmente la survie de cellules NSCLC au CisPt. Ces deux isoformes sont engagées dans des fractions polysomiques légères. Des analyses de bases de données de Ribo-seq et de MS ont révélé l’existence de sORF dans ces deux isoformes. Par transfection de vecteurs contenant ces isoformes 5’UTR-IPA et en balisant leurs sORF, j’ai observé en ImmunoFluorescence (IF) et Western Blot que l’isoforme 5’UTR-IPA de PRKAR1B code une miP. La délétion de cette isoforme IPA endogène ou la mutation de l’ATG de sa sORF par CRISPR ont donné un phénotype similaire au siARN. Il s’agit de la première isoforme 5’UTR-IPA codant une miP (miP-5’UTR-IPA). Par croisement de nos données de 3’-seq avec des données de Ribo-seq et de MS, nous avons identifié une centaine d’isoformes miP-5’UTR-IPA potentielles induites par le CisPt. Deuxièmement, je me suis intéressé à la possibilité de sensibiliser des cellules NSCLC au CisPt en ciblant U1 snRNP par un oligonucléotide (U1-AMO) qui induit l’IPA dans de nombreux gènes. Dans plusieurs lignées cellulaires NSCLC, j’ai pu montrer une sensibilisation par U1-AMO au CisPt en termes d’inhibition de croissance cellulaire et d’induction de dommages à l’ADN (foyers ƴH2AX). Cette sensibilisation est liée à une réduction (en 3’-seq et RT-qPCR) de l'expression des ARNm canoniques dans des gènes de réparation des pontages de l'ADN (voies de Fanconi et de l’excision de nucléotides). Cependant, U1-AMO prévient les blocages de cycle cellulaire induits par le CisPt, ainsi que les effets du CisPt sur le ratio IPA:LE de nombreux gènes. Mes travaux montrent l’impact de l’IPA sur la réponse des cellules cancéreuses au CisPt, et révèlent un nouveau paradigme génétique, appelé miP-5’UTR-IPA, dans lequel des gènes produisent par IPA des transcrits courts codant des miP
During studies on alternative polyadenylation (APA), short transcripts ending in an alternative last exon were discovered, known as intronic polyadenylation (IPA). IPA is regulated by splicing factors (including U1 snRNP), polyadenylation factors and transcription elongation factors (including CDK12). IPA isoforms are regulated by genotoxic agents (inducing DNA damage), including UV radiation and doxorubicin. Conversely, CDK12 inhibitors increase both IPA in DNA repair genes and cellular sensitivity to genotoxic agents. IPA often occurs in the coding region of genes, generating carboxy-terminally altered proteins. However, IPA transcripts are also generated in the first introns of genes, known as 5'IPA. Many 5'IPA transcripts are degraded by the nuclear exosome, but some are abundant and have a low coding potential. Two of these, derived from the ASCC3 and CDKN1A genes, have non-coding functions. In addition, studies using Ribo-seq and mass spectrometry (MS) are showing the existence -in mRNAs and lncRNAs- of small open reading frames (sORF) encoding microproteins (miP, proteins of less than 100 aa) which can be functional. No miP has been reported in 5'IPA isoforms. Cisplatin (CisPt) is a DNA-crosslinking agent widely used in non-small cell lung cancer (NSCLC). My team observed, by 3'-seq in NSCLC cells, that CisPt increases the expression of IPA isoforms compared to canonical mRNAs (IPA:LE ratio) in many genes, and that some IPA isoforms are poorly associated with heavy polysomes and are derived from the region upstream of the annotated translation initiation site of the gene (5'UTR-IPA isoforms). My objectives were to determine the role of IPA in cell response to CisPt. I first looked at the role of 5'UTR-IPA isoforms. For two of them, derived from the PRKAR1B and PHF20 genes, I showed that their depletion by siRNA increased the survival of NSCLC cells to CisPt. These two isoforms are associated with light polysomal fractions. Analyses of Ribo-seq and MS databases revealed the existence of sORFs in these two isoforms. By transfecting vectors containing these 5'UTR-IPA isoforms and by tagging their sORFs, I observed by ImmunoFluorescence (IF) and Western Blot that the 5'UTR-IPA isoform of PRKAR1B encodes a miP. Deletion of this IPA isoform or mutation of the sORF ATG endogenously by CRISPR gave a phenotype similar to the siRNAs. This is the first 5'UTR-IPA isoform encoding a miP (miP-5'UTR-IPA). By cross-referencing our 3'-seq data with Ribo-seq and MS data, we identified around a hundred potential miP-5'UTR-IPA isoforms induced by CisPt. Secondly, I investigated the possibility of sensitizing NSCLC cells to CisPt by targeting U1 snRNP with an antisense oligonucleotide (U1-AMO), that induces IPA in many genes. In several NSCLC cell lines, I showed sensitization to CisPt by U1-AMO in terms of cell growth inhibition and DNA damage induction (ƴH2AX foci). This sensitization is linked to a reduced expression of the canonical mRNAs of DNA crosslinks repair pathways (Fanconi and nucleotide excision repair), as shown by 3'-seq and RT-qPCR. However, U1-AMO prevented CisPt- induced cell cycle block and the effects of CisPt on the IPA:LE ratio of many genes. My work shows the impact of IPA on the response of cancer cells to CisPt, and reveals a new genetic paradigm, called miP-5'UTR-IPA, in which genes produce short miP-encoding transcripts by IPA

La maturation des ARN implique un grand nombre d’évènements post-transcriptionnels, parmi lesquels la polyadénylation qui constitue une étape clé. Chez Arabidopsis, la présence de l’hétérochromatine au niveau des introns de certains gènes peut influencer considérablement la polyadénylation de leur transcrits. INCREASED IN BONSAI METHYLATION2 (IBM2) est une protéinequi contrôle cette catégorie de gènes en reconnaissant l’hétérochromatine au niveau des introns via son domaine BOMO-ADJACENT HOMOLOGY (BAH). IBM2 se lie à l’ARNm par son motif RNA RECOGNOTION (RRM), afin d’assurer la transcription complète de ces gènes cibles en favorisant l’utilisation d’un site distal de polyadénylation. Par conséquent, en mutant IBM2, des plus transcrits courts sont synthétisés suite à une polyadénylation précoce au niveau de la régionhétérochromatique. Durant ma thèse, j’ai cherché à comprendre les mécanismes moléculaires sous-jacents de cette régulation tout en étudiant le rôle du complexe protéique IBM2. Nous avons identifié des protéines partenaires d’IBM2 déjà étudiées telle que ENHANCED DOWNY MILDEW2 (EDM2) et ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), ainsi qu’une nouvelle protéine interagissant physiquement avec IBM2 et d’autres protéines. La mutation du gène correspondant à cette protéine conduit à une réduction de l’expression globale des cibles d’IBM2testées, accompagnée d’un niveau réduit de transcrits longs fonctionnels. Moyennant un crible génétique des suppresseurs de la mutation ibm2, nous avons identifié plusieurs facteurs agissant en amont de la voie IBM2, notamment la protéine FLOWERING TIME CONTROL (FPA). FPA est une protéine capable de s’associer à l’ARN pour favoriser l’utilisation de sites proximaux de polyadénylation de plusieurs gènes cibles, avec parmi eux des gènes contrôlés par IBM2, ce qui suggère que la transcription complète de ces gènes dépend étroitement des actions antagonistes entre IBM2 et FPA. Nos résultats ont montré que le choix du site de polyadénylation de gènes contenant de l’hétérochromatine dépend de plusieurs protéines agissant en différents complexes ainsi que l’interconnexion avec d’autres voies
RNA maturation implies numerous post-transcriptional modifications in whichpolyadenylation is a key step. In Arabidopsis, the heterochromatin found within introns(intronic-HC) can impact transcripts polyadenylation of host genes. INCREASED IN BONSAI METHYLATION2 (IBM2), an RNA-binding protein containing a bromo-adjacent homology (BAH) domain, interacts with intronic-HC to produce functional full-length transcripts by promoting distal polyadenylation. Loss of IBM2 function triggers short transcripts production due to premature polyadenylation from the heterochromatic region. During my thesis, I investigated the role of proteins that may belong to different sub-complexes in the regulation of intronic-HC containing genes. We identified IBM2 partners, including ENHANCED DOWNY MILDEW 2 (EDM2) and ASI-IMMUNOPRECIPITATED PROTEIN1 (AIPP1), and a novel partner that interacts directly with IBM2 and other proteins. Mutating the corresponding gene of the novel partner results in decreased expression of tested IBM2-targets such as IBM1 encoding an H3K9demethylase and the disease resistance gene RECOGNITION OF PERONOSPORA PARASITICA 7 (RPP7), accompanied with compromised use of their distal polyadenylation sites. By conducting a genetic screen of ibm2 mutation suppressors, we identified factors belonging to different pathways that act upstream of IBM2, among them the FLOWERING TIME CONTROL PROTEIN (FPA). FPA is an RNA-binding protein that promotes the use of proximal polyadenylation sitesof several genes such as IBM1. Our data bring evidence that antagonistic actions of FPA and IBM2 regulates polyadenylation sites choice at intronic-HC containing genes. These results provide new insights to understand the interplay between heterochromatin and RNA processing

Cancer and inflammation are connected by intrinsic pathways and extrinsic pathway where the intrinsic pathway is activated by genetic events including mutation, chromosomal rearrangement or amplification, and the inactivation of tumor-suppressor genes, as well as the extrinsic pathway, is the inflammatory or infectious conditions that increase the cancer risk. On the other hand, introns are non-coding elements of the genome and play a functional role to generate more gene products through splicing out, transcription, polyadenylation, mRNA export, and translation. Moreover, introns also may act as a primary element of some of the most highly expressed genes in the genome. Intron may contain their regulatory function as CRISPR system which is activated after the demand of specific gene for specific protein formation where those are required for gene expression, they go for transcription and rest of them form splicing. This chapter will focus on the plausible role of introns to influence the genetic events of inflammation-mediated cancer cell development.

Abstract In eukaryotes, pre-mRNA is bound by protein either simultaneously with the initiation of transcription or very soon thereafter (1-3), and it is not possible to isolate pre-mRNA molecules free from a unique set of abundant nuclear proteins unless protein denaturants are used in the purification scheme. The packaging of DNA into nucleosomes, and their association in the 30 nm chromatin fibre, greatly foreshortens the DNA substrate and provides some protection of the genome from nuclease activity (4). However, the major pre-mRNA binding proteins possess well- characterized, helix-unwinding activities (5-8) and, in comparison with nucleosome packaging, provide less protection from nuclease activity (9,10). It seems then, that the pre-mRNA packaging mechanism functions primarily to maintain the transcript in a single-stranded state. This generally fits with the short-lived nature of pre- mRNA, and with the fact that nascent transcripts must be accessed by factors involved in intron removal, polyadenylation, and transport. Unlike rRNA, tRNA, and snRNA, pre-mRNAs are usually transcribed from single-copy genes and the pool of total nuclear pre-mRNA reflects great length heterogeneity (hence the term, heterogeneous nuclear RNA or hnRNA).

Abstract Pre-mRNA transcripts must be processed and transported to the cytoplasm where the mature mRNAs are translated into proteins. For most RNA polymerase II tran-scripts, this processing includes addition of a 7-methyl-guanosine cap structure at the 5’ end of the nascent RNA transcripts; hnRNP assembly; removal of non-coding intron regions and ligation of exons; 3’ end cleavage and polyadenylation; and the ex¬ change of hnRNP proteins for mRNP proteins. Splicing occurs in a multicomponent complex termed a spliceosome. Many of the detailed biochemical steps involved in the prc-mRNA splicing reaction have been extensively studied in vitro and are well understood (for reviews sec Chapters 3-8). However, the integration of the splicing reaction with other related cellular functions, such as transcription and RNA trans-port, is best studied in intact cells. The cellular approach to understanding the organization of nuclear function has lagged somewhat behind the in vitro approach. However, during the past few years, major inroads have been made toward our understanding of where in the nucleus pre-mRNA splicing occurs and how the organization of both splicing factors and substrates are interrelated to the transcrip¬ tional activity of the cell. In this chapter, the nuclear organization of both snRNP and non-snRNP RNA processing factors, as well as pre-mRNA substrates, arc examined. In addition, their organization throughout the cell cycle, and the relationship of their nuclear distribution to the transcriptional activity of the cell, are discussed.

Abstract Normal development of human tissues requires the activation of specific genes during various embryonic stages so that stage-specific protein isoforms and other gene products appear in their proper developmental windows. While transcriptional initiation starts the gene expression cascade, nascent transcripts must be modified and the mature RNA products correctly localized within the cell. Most of the nuclear processing machinery is devoted toward producing the major RNAs required for the translational apparatus (Fig. 125–1). Pre-messenger RNAs (pre-mRNAs) are transcribed by RNA polymerase II (Pol II) and modified (capped) at the 5′ end by 7-methylguanosine, introns are removed by the spliceosome and the mature 3′ ends of mRNAs are generated by endonucleolytic cleavage which is generally followed by polyadenylation. Ribosomes begin life in the nucleolus where pre-ribosomal RNA (pre-rRNA) processing involves a series of nucleolytic cleavage steps as well as nucleotide modifications which results the synthesis of subunit precursors. These precursors are subsequently exported into the cytoplasm for the final modification steps required to form functional subunits. A similar biogenesis pathway is followed by pre-transfer RNAs (pre-tRNAs) although the majority of these processing events occur outside of the nucleolus. Overall, the cell expends an enormous amount of metabolic energy on these RNA processing and localization pathways. Recent estimates suggest that the spliceosome alone may be composed of hundreds of protein factors in addition to the five small nuclear RNAs (snRNAs) (Jurica and Moore, 2003). Mutations in any one of these factors, or the cis-acting elements to which they bind, may result in human disease.

Abstract All three major classes of RNA undergo post-transcriptional processing by nucleolytic cleavage and base modification before they become functional (1). A general theme in RNA processing is (cis or trans) RNA–RNA interactions and the association with a macromolecular complex. Ribosomal RNA precursors are processed by a series of endonucleolytic cleavages that are catalyzed by RNA or protein enzymes. The rRNA bases are modified at sites which are guided by base pairing with small nucleolar (sno) RNAs (2). Similarly, tRNAs are cleaved from precursors, and the site of the 5’ cleavage is guided by bases pairing with the RNA component of RNase P. In addition, many bases of tRNA are modified by a variety of enzymes that select the base by RNA–RNA interactions that result from the tRNA structure (3). The mRNA precursors also undergo a variety of processing reactions. These include cleavage, 5’ capping, 3’ polyadenylation and intron excision (splicing). An important aspect of RNA splicing is that it produces informationally correct mature mRNA and, indeed, the appropriate mRNA version in the case of alternative splicing. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that is composed of numerous proteins and several small RNAs that base pair with the mRNA and guide the sites of catalysis. Indeed, spliceosome RNAs may catalyze some of the steps of splicing. More recently, it was found that mRNAs undergo other forms of RNA processing that are collectively termed RNA editing since they alter the sequence from that encoded in the structural gene (4). RNA editing falls into two categories: base modification and nucleotide insertion/deletion. The first observed, and perhaps most complex, form of RNA editing is that which occurs in the mitochondrion of kinetoplastid protozoa (trypanosomatids). This type of editing is the subject of this chapter and has been covered in several excellent previous reviews (5, 6). This type of editing inserts and deletes uridylates (Us) to produce mature mRNAs with sequences that are specified by small guide RNAs (gRNAs). The editing in trypanosomes occurs in association with a macromolecular complex. It occurs in all kinetoplastids but is most extensive in African trypanosomes. Editing in this group will be the focus of this review.

The endothelium plays a crucial role in the regulation of the fibrinolytic process, since it synthesizes and secretes tissue-type plasminogen activator (t-PA) as well as the fast-acting plasminogen activator inhibitor (PAI-1). Molecular cloning of full-length PAI-1 cDNA, employing a human endothelial cDNA expression library, and a subsequent determination of the complete nucleotide sequence, allowed a prediction of the amino-acid sequence of the PAI-1 glycoprotein. It was observed that the amino-acid sequence is significantly homologous to those of members of the serine protease inhibitor ("Serpin") family, e.g. αl-antitrypsin and antithrombin III. Serpins are regulators of various processes, such as coagulation, inflammatory reactions, complement activation and share a common functional principle and a similar structure, indicative for a common primordial gene. The intron-exon arrangement of Serpin genes may provide a record for the structure of a primordial gene. A comparison of the location of introns among members of the Serpin family reveals that some introns are indeed present at identical or almost identical positions, however in many other cases there is no correspondence between the intron positions among different Serpin genes.Obviously, more data on the chromosomal gene structure of members of this family are required to formulate a scheme for the evolutionary creation of the Serpins. To that end, we have established the number and the precise location of the introns in the PAI-1 gene and have compared these data with those reported on other Serpin genes. For that purpose a human genomic cosmid DNA library of about 340.000 independent colonies was screened with radiolabelled full-length PAI-1 cDNA as probe. Two clones were found which contain the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes and nucleotide sequence analysis demonstrate that the PAI-1 gene comprises about 12.2kilo basepairs and consists of nine exons and eight introns. Intron-exon boundaries are all in accord with the "GT-AG" rule, including a cryptic acceptor splice site found in intron 7. Furthermore, it is observed that intron 3 of the PAI-1 gene occupies an identical position as intron E of chicken ovalbumin and intron E of the ovalbumin-related gene Y. The location of the other seven introns is unrelated to the known location of introns in the genes encoding the Serpins, rat angiotensin, chicken ovalbumin (and gene Y), human antithrombin III and human al-antitrypsin. The 3' untranslated region of the PAI-1 gene is devoid of introns, indicating that the two mRNA species detected in cultured endothelial cells which share an identical 5' untranslated segment and codogenic region, but differ in the length of the 3' untranslated region, arise by alternative polyadenylation. An extrapolation of the position of the introns to the amino-acid sequence of PAI-1, and adaption of the view that the subdomain structure of the Serpins is analogous, shows that the introns of PAI-1 are non-randomly distributed. Except for intron 7, the position of the other seven introns corresponds with randon-coil regions of the protein or with the borders of β-sheets and a-helices. Extrapolation of the position of introns in the genes of other Serpins to their respective amino-acid sequences and subdomain structures also reveals a preference for random-coil regions and borders of subdomains. These observations are reminiscent of an evolutionary model, called "intron sliding", that accounts for variations in surface loops of the same protein in different species by aberrant splicing (Craik et al., Science 220 (1983) 1125). The preferential presence of introns in gene segments, encoding these variable regions, and absence in regions determining the general folding of these proteins would explain conservation of the structure during the evolution of those genes.

Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.