Pre-mRNA splicing and other RNA processing events occur co-transcriptionally. High resolution kinetic studies performed in our lab showed splicing-dependent RNA Pol II (RNA polymerase II) pausing near the 3’ splice site of a reporter gene. Pausing requires splicing, as mutations that block splicing lead to loss of pausing, and restoring splicing restores pausing. It was proposed that RNA Pol II pausing may occur at splicing-dependent transcriptional checkpoints. In this study, I aimed to search for splicing helicases that might couple splicing with transcription. The ts alleles prp5-1 and prp16-2 were found to cause transcription defects. These genes encode RNA helicases that were reported to act as fidelity factors during splicing. In vivo RNA labelling and RT-qPCR experiments performed with these temperature-sensitive mutants demonstrated reduced transcription coinciding with the splicing defect at restrictive temperature. Furthermore, RNA Pol II ChIP analysis showed polymerase accumulating over intron-containing genes in both mutants. ChIP analysis using antibodies specific to the phosphorylation status of the CTD (Carboxy Terminal Domain) of RNA Pol II, revealed that the apparently stalled polymerase is hyper-phosphorylated at serine 5. Intriguingly, prp8-R1753K, a ts allele of PRP8, a non-helicase splicing factor mutant also showed reduced nascent RNA synthesis but no RNA Pol II accumulation. To elucidate the reason for the observed RNA Pol II accumulation and to identify a possible splicing-dependent transcriptional checkpoint factor, prp5-1 was investigated further. RNA Pol II ChIP-Seq analysis verified that maximum enrichment genome-wide occurred on introns at restrictive conditions in prp5-1, supporting the earlier observation. Furthermore, the double mutant strain cus2Δprp5-1 abolished the RNA Pol II accumulation observed in prp5-1 at restrictive temperature and restored transcription. Recreating a stalled spliceosome in a U2 mutant strain also showed RNA Pol II accumulation in the presence of Cus2p, as observed in prp5-1. My observations suggest a link between transcription and monitoring of splicing and indicate that Cus2p, a U2 snRNP associated protein, could be a checkpoint factor in transcription prior to pre-spliceosome formation. I speculate that fidelity factors may impose transcriptional checkpoints at different stages of splicing.

The spliceosomal U5 snRNP contains several proteins with well characterised functions in splicing, including: Brr2, an ATPase/RNA helicase that disrupts U4/U6 and U2/U6 snRNA base pairing during activation of the spliceosome; Snu114, a GTPase that controls the action of Brr2; and Prp8, the largest and most conserved protein considered to have a central role in the spliceosome, which interacts directly with Snu114 and Brr2. Yeast Cwc21 is one of twelve Bact complex proteins that associate with spliceosomes shortly before the first step of splicing catalysis. Cwc21 interacts directly with Prp8 and Snu114, as does its human orthologue, the SR protein SRm300/SRRM2. Although, Cwc21 is not essential for yeast cell viability, it is required for sporulation. This work aims to identify the function of Cwc21 during meiosis. PP1 is a protein phosphatase required for both steps of splicing. Multiple sequence alignments of Snu114 and Prp8 revealed the presence of putative PP1 binding motifs that are well conserved among different species. This led me to hypothesize that PP1 may interact with Snu114 and/or Prp8 to regulate these or other interacting proteins. By screening intron-containing genes that are expressed in meiosis, I found that Cwc21 is required for splicing HRB1 transcripts. In addition, I show that HRB1 is also required during meiosis. The HRB1 intron contains an unusual branchsite sequence, TACTAATG, which when changed to the consensus branchsite sequence restores sporulation in the absence of Cwc21. Therefore, it is likely that Cwc21 promotes the expression of HRB1 during an early stage of meiosis by stabilising its pre-mRNA in the catalytic centre of the spliceosome. This study demonstrates a novel function for Cwc21 during meiosis. Using yeast two hybrid assay I have identified the interacting regions of Cwc21, PP1 and Brr2 in Snu114. Through biochemical studies I provide evidence for mutually exclusive interaction of Cwc21 and PP1 in the putative PP1 binding motif situated in Snu114 domain ‘IVa’. In the case of yeast Snu114, the PP1 binding motif has a novel sequence ‘YGVQYK’. I also show that the affinity of Cwc21 and PP1 for Snu114 is influenced by the different nucleotide-bound states of Snu114. Furthermore, I show that mutations in Snu114 domain ‘IVa’ restrict Snu114 function during meiosis and affect the MER1 splicing regulatory network. Therefore, Snu114 may play a role in modulating the conformational state of the catalytic spliceosome through its interactions with Cwc21/PP1 in regulating subsets of genes during meiosis. Finally, I show that PP1 is a putative regulator of Prp8.

The RNA helicase Prp43p is one of the few members of the DEAH-box helicase family that is known to operate in more than one cellular process in Saccharomyces cerevisiae. With roles in ribosome biogenesis and pre-mRNA splicing, Prp43p may be important in maintaining a communication conduit between these two pathways. Our studies provide insights into how Prp43p function is regulated through the use of three cofactors, Ntr1p, Pfa1p, and Gno1p, all of which interact with Prp43p at different steps of pre-mRNA splicing or ribosome biogenesis. Each cofactor contains a unique G-patch domain and our data show that they associate with Prp43p in a mutually exclusive manner. A strong growth defect and RNA processing phenotypes are seen upon overexpression of Pfa1p due to the dominance of Pfa1p interaction with Prp43p. Moreover, excess Pfa1p precludes Prp43p from interacting with either 35S pre-rRNA or U6 snRNA, indicating this one cofactor can negatively regulate Prp43p recruitment into ribosome biogenesis and pre-mRNA splicing pathways, respectively. We have determined that Ntr1p and Gno1p are able to compete with one another for Prp43p occupancy. Similar to Ntr1p, we show that the G-patch domain of Gno1p contributes to its association with Prp43p. To further understand pathway specificity of Prp43p, we characterized conditional prp43 alleles with mutations C-terminal to the conserved RecA domains of Prp43p. These novel alleles affect pre-mRNA splicing and ribosome biogenesis, though none are mutually exclusive. Multiple prp43 alleles are deficient in tri-snRNP formation, a previously uncharacterized phenotype in prp43 mutants. The majority of our prp43 mutants display varying rRNA defects, with some alleles impacting ribosome biogenesis more severely or moderately than known prp43 ATPase mutants. To correlate the processing defects seen in each allele, we have determined the extent of association of the mutants with each G-patch protein. Altogether, our data support a working model for Prp43p in which its substrate specificity, activation, and cellular distribution is coordinated through the efforts of the three G-patch proteins in yeast and sheds light on potential mechanisms of general DExH/D helicase function and regulation. / text

No description available.

No description available.

Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.

In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today. In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP? Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process. In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription. / Bei Säugetieren wird ein großer Teil des Genoms als nicht-kodierende RNAs transkribiert. Es gibt immer mehr Hinweise darauf, dass nicht-kodierende RNAs eine wichtige Rolle sowohl für die normale Zellfunktion als auch bei Krankheitsprozessen wie Krebs oder Neurodegeneration spielen. Die Interpretation der Funktionen nicht-kodierender RNAs und der molekularen Mechanismen, über die sie wirken, ist eine der wichtigsten Herausforderungen, denen die RNA-Biologie heute gegenübersteht. In meiner Promotionsarbeit habe ich die Rolle von 7SK, einer der am häufigsten vorkommenden nicht-kodierenden RNAs, bei der Entwicklung und Funktion von Motoneuronen untersucht. 7SK ist eine RNA, die aus 331 Nukleotiden besteht und deren Struktur bekannt ist. Sie wird von der RNA-Polymerase III transkribiert. Sie bildet vier Stem-Loop (SL)-Strukturen, die als Bindungsstellen für verschiedene Proteine dienen. LARP7 bindet an SL4 und schützt das 3'-Ende vor exonukleolytischem Abbau. SL1 dient als Bindungsstelle für HEXIM1, das den P-TEFb-Komplex rekrutiert, der aus CDK9 und Cyclin T1 besteht. P-TEFb hat eine stimulierende Rolle für die Transkription und wird durch Sequestrierung durch 7SK reguliert. In jüngerer Zeit wurde eine Reihe von heterogenen nukleären Ribonukleoproteinen (hnRNPs) als 7SK-Interaktoren identifiziert. Eines davon ist hnRNP R, von dem gezeigt wurde, dass es eine Rolle bei der Entwicklung von Motoneuronen spielt, indem es das Axonwachstum reguliert. Durch die Interaktion mit P-TEFb und RNA-bindenden Proteinen kann 7SK als Drehscheibe für die Rekrutierung und Freisetzung verschiedener Proteine betrachtet werden. Die Fragen, mit denen ich mich während meiner Doktorarbeit beschäftigt habe, lauten wie folgt: 1) Welche Region von 7SK interagiert mit hnRNP R, einem Hauptinteraktor von 7SK? 2) Welche Effekte treten in Motoneuronen auf, wenn die Bindung von hnRNP R an 7SK inhibiert wird? 3) Gibt es zusätzliche 7SK-bindende Proteine, die die Funktionen des 7SK RNPs regulieren? Mit Hilfe von in vitro und in vivo Experimenten fand ich heraus, dass hnRNP R sowohl die SL1- als auch die SL3-Region von 7SK bindet, und dass P-TEFb nach der Deletion der SL1-Region nicht rekrutiert werden kann, aber in der Lage ist, an eine 7SK-Mutante mit Deletion von SL3 zu binden. Um die Frage zu beantworten, wie sich die 7SK-Mutationen auf Axonwachstum in primären Motoneuronen der Maus auswirken, führten wir Rettungsexperimente an Motoneuronen unter Verwendung lentiviraler Vektoren durch. Die Konstrukte wurden so konzipiert, dass sie 7SK-Deletionsmutanten durch den U6-Promotor der Maus exprimieren und gleichzeitig eine 7SK-shRNA von einem H1-Promotor für die Depletion von endogenem 7SK transkribieren. Mit diesem System fanden wir heraus, dass 7SK-Mutanten, die Deletionen von SL1 oder SL3 beherbergen, den Axon-Wachstumsdefekt von 7SK-depletierten Motoneuronen nicht retten konnten, was darauf hindeutet, dass 7SK/hnRNP R-Komplexe für diesen Prozess von Bedeutung sind. Um neue 7SK-Bindungsproteine zu identifizieren und ihre Funktionen zu untersuchen, führte ich Pulldown-Experimente durch, bei denen ich ein biotinyliertes RNA-Antisense-Oligonukleotid verwendete, das an die U17-C33-Region von 7SK bindet und dadurch Aufreinigung endogener 7SK-Komplexe erlaubt. Nach der Massenspektrometrie der gereinigten 7SK-Komplexe identifizierten wir eine Reihe neuer 7SK-Interaktoren. Einer davon ist der Smn-Komplex. Ein Mangel des Smn-Komplexes verursacht die Motoneuronerkrankung Spinale Muskelatrophie (SMA), die durch den Verlust der unteren Motoneuronen im Rückenmark gekennzeichnet ist. Es wurde bereits gezeigt, dass Smn mit hnRNP R interagiert. Dementsprechend fanden wir Smn als Teil des 7SK/hnRNP R-Komplexes. Diese Proteom-Daten deuten darauf hin, dass 7SK neben der Transkription möglicherweise auch in anderen Signalwegen wie der spliceosomalen snRNP Biogenese eine wichtige Rolle spielt.

No description available.

Splicing is a process, during which non-coding sequences (introns) are cleaved out of pre-mRNA, and exons are ligated. This whole process is catalysed by a multi-megadalton splicing complex, composed of five small nuclear ribonucleoprotein particles (shortly snRNPs), which each contains its own small nuclear RNA molecule and specific set of proteins. During the biogenesis of snRNPs, U4 and U6 snRNPs are assembled to form the di-snRNP, which further associates with U5 snRNP and gives rise to tri-snRNP. With the help of mass spectrometry, we have found previously uncharacterized protein interacting with U5 snRNP, called TSSC4. By immunoprecipitation, I confirmed TSSC4 as a U5 snRNP specific protein and identified the region of TSSC4 responsible for interaction with U5 snRNP. I also showed that TSSC4 interacts with PRPF19, a component of complex driving the catalytic activation of the spliceosome and that this interaction is U5 snRNP-independent. Knockdown of TSSC4 in HeLa cells results in accumulation of di-snRNAs and U5 snRNP in Cajal bodies, nuclear compartments involved in snRNP biogenesis. Similar phenotype was previously observed upon inhibition of tri-snRNP assembly. To analyse the importance of TSSC4 for tri-snRNP assembly, I separated individual snRNPs by glycerol gradient ultracentrifugation...

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