Heidelberg, University, Diss., 2002. / Dateien im PDF-Format.

Zugl.: Göttingen, Univ., Diss., 2009

Zugl.: Göttingen, Univ., Diss., 2009

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.

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.

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.

No description available.

It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.