Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

No description available.

No description available.

No description available.

Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing. Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product. We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8. Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions. It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites. U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1. hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner. HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell. We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent. Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.