The presence of introns in all the eukaryotic genomes identified so far underscores the fundamental and ubiquitous role of pre-mRNA splicing. The spliceosomal machinery, comprised of five small nuclear RNAs and several protein factors, catalyzes the two-transesterification reactions of splicing with precision and consistency. Through a complex network of protein-protein and RNA-protein interactions it ensures the removal of the intron and ligation of the flanking exons to yield the mature mRNA.
Prpl7 is a splicing factor that functions at the second-step of splicing (Vijayraghavan et all, 1989). Null alleles of prpl7 are viable at 23°C but die at temperatures above 33°C (Jones et al.9 1995). Besides its functions in pre-mRNA splicing, mutants in PRP17ICDC40 were independently shown to affect cell-cycle progression, particularly the Gl/S and G2/M transitions (Chawla et a/., 2003). In this study, we have attempted a further characterization of Prpl7 to analyze both its role in pre-mRNA splicing and in cell-cycle progression with an aim to decipher underlying reasons for the interlinking of these two cellular processes. Different experimental approaches were adopted to achieve this goal. Global gene-expression profiling provided an overview of all the transcripts affected in a prpl 7 mutant and allowed its comparison with mutants of other splicing factors. This exercise aided in identification of both pre-mRNA splicing and cell-cycle related effects of Prpl7. Biochemical analysis of the Prpl7 spliceosomal associations have provided further clarity on the part played by Prpl7 in pre-mRNA splicing. A genome-wide two-Hybrid screen for interacting partners of Prpl7 was undertaken and uncovered two Likely interacting partners of Prpl7.
Global expression profiling of splicing mutants
Pleiotropic phenotypes observed in mutants of prpl 7 and few other splicing factors have been speculated to arise from either the multi functionality of the factor or more likely due to a specific requirement of the factor in splicing of a select subset of transcripts, that encode proteins essential to the affected cellular pathway. These observations raise questions about the ubiquitous requirement of factors in pre-mRNA splicing. To understand these aspects of splicing, we studied the effects of splicing factor mutants on a genome-wide scale. Using splicing-sensitive DNA microarrays imprinted with all yeast ORFs and in addition, independent spots for a majority of the intron sequences, we analyzed the global expression changes triggered by the inactivation of temperature-sensitive mutations in PRP17 or PRP22.
Experiments with prp2-l mutant strain detect, as expected, an increase in pre-mRNA levels at the intron spots and further demonstrated that the ORF spots detect a decrease in mRNA levels in these DNA microarrays. These results established the DNA micro arrays as tools for the analysis of splicing on a global scale. The temporal alterations in transcript profiles in prpl 7 and prp22 mutants, as compared to the wild type, revealed both shared and unique effects of these factors on clusters of intron-containing transcripts. Such differential effects, on intron-containing transcripts, amongst the splicing mutants implicate specialized roles for each of these factors. Through analysis of the set of intron-containing transcripts affected in
prpl7Δ cells, we infer those attributes of these pre-mRNA substrates, which predispose a need for Prpl 7. We find that splicing of introns longer than 200nts has a stronger dependence on Prpl7. The distance between consensus intron elements- the branch-nucleotide and the 3'splice-site (B), also imposes a requirement for Prpl7. Introns with a 13nts or lesser distance between these elements are spliced even in the absence of Prpl 7, both in vivo and in vitro. The 5'splice-site to branch-nucleotide distance (A) also influences the need for Prpl7. Most introns with a A/B ratio of less than 2 undergo Prpl7 independent splicing in vivo.
Intron-containing genes that could be responsible for the pleiotropic phenotypes of prpl7 were also identified through the global splicing analysis. These included splicing targets that act at the Gl-S phase such as ANC1/TAF14, TMD4, PHO85 and those at the G2-M phase of the cell-cycle; TUB], TUB3, GIM5, MOBl UBC9. Recently, a different study implicates ANC1ITAF14 as the intron-containing gene responsible for the cell-cycle phenotype associated with prpl7 (Dahan and Kupiec, 2004). Our global analysis of all intron-containing transcripts with compromised expression in prpl7A cells identify, in addition, PHO85 as a possible regulator underlying cell-cycle effects in this mutant. Pho85 is a cyclin-dependent kinase that functions at both the Gl/S and M/Gl phases of the division cycle (Moffate* al., 2000). Synergistic growth defects in double mutants of prpl7 and pho85 have uncovered a novel role for Prpl7 in bud morphogenesis. Our micro array data also reveals compromised expression levels for several key intronless cell-cycle rregulatory genes indicating a possible splicing-independent role for Prpl7 in the cell-cycle. Examples of such transcripts are: the Gl cyclins CLN1, CLN2 and CLN3; CDC6, required for assembly of the pre-replication complex at sites of replication origin; and the cell-cycle regulatory transcription factors: SWI5 and ACE2. The global analysis has therefore enabled, for the first time, a characterization of the splicing substrate specificity of Prpl7 and has also uncovered the effects of this protein on gene expression during cell-cycle progression (Fig. V.I A).
Spliceosomal interactions of Prpl7
To understand the function and associations of Prpl7 in the spliceosome, we have examined its snRNP interactions and determined the time point of its coalescence on assembling spliceosomes. A functional epitope tagged-Prpl7 was created using the polyoma middle T-antigen and the poly-HIS tags (Stevens et aln 1999). Through immunoprecipitation analyses performed with splicing extracts, from this strain, we find Prpl7 to associate with three spliceosomal snRNPs- U2, U5 and U6, implicating an interaction with active spliceosomes or post-splicing complexes. Specific biochemical depletion of any one of these snRNAs, through oligo-directed RNaseH cleavage, did not have a drastic effect on the association of Prpl7 with the other two snRNAs. To decipher the point at which Prp 17 joins the assembling spliceosomes, we examined the presence of Prp 17 in in vitro assembled complexes generated under various conditions. The conditions adopted were designed to stall and enrich for •assembly intermediates. A co-immunoprecipitation of the input precursor RNA and reaction intermediates revealed an early association of Prp 17 with the assembling Spliceosome prior to its catalytic activation. This association occurred in the A2-1 complex, which contains the U4/U6.U5 tri-snRNP along with the Ul and U2snRNPs. Prpl7 was found to associate with all subsequent complexes until the completion of catalytic transesterification reactions and possibly continue with the spliced-out introns complex (Fig. V.1B).
Identification of two novel interacting partners of Prpl7 from a genome-wide two-hybrid screen
Although several genetic interacting partners of PRP17 are known, none display a direct physical association with Prpl7. Knowledge of the proteins that Prpl7 interacts with can further the functional characterization of this protein and aid in deciphering its link to cell-cycle progression. A genome-wide screen for interacting partners using Prpl7 as bait was carried out in a two-hybrid system with a yeast genomic DNA-B42 activation-domain library (Gyuris et al., 1993). Through this screen we identified two interacting partners of Prpl 7- YOL078W, an essential gene and SGML The domain in the 1176 amino acid YOL078W protein responsible for interaction with Prpl7 was mapped to a 225 amino acid segment in the C-terminai region of this protein. The N-terminal region of the protein appears to exert a negative effect on the interaction with Prpl7. While YOL078w does not have any apparent role in pre-mRNA splicing, a majority of the cells arrest with small buds indicating a late Gl or early S phase arrest upon transcriptional shut-down of YOL078W. YOL078W has been independently characterized as AVOl, a component of the TOR complex, involved in nutrient sensing and cell size regulation (Loewith et al, 2002). Other reports show it tto be a component of a complex that interacts with Ceglp, a nuclear protein involved in mRNA capping (Gavin et al, 2002). We hypothesize that Prpl7 and Avol may exist in a dynamic nucleocytoplasmic complex possibly functioning in either cell-cycle regulation, RNA processing or both. Through this study we have
Established the use of splicing-sensitive microarrays as tools for the characterization of pre-mRNA splicing factors. Simultaneous assessment of the
effects on other cellular pathways was accomplished through expression profiling
of all the intron-containing and intronless genes.
Deciphered the differential dependence of pre-mRNA substrates on spliceosome
factors at a global scale.
Predicted the substrate-specificity of the second-step splicing factor, Prpl7, and
verified some of these predictions in vitro.
Gathered evidence for a possible splicing-independent effect of Prpl7 on the cell
division cycle.
Uncovered a novel function of Prpl7 in bud morphogenesis, as deduced from its
synergistic genetic interaction with PHO85.
Identified U2, U5 and U6 snRNPs as interacting partners of Prpl7 in both xtracts
and in in vitro splicing reactions.
Determined the point of coalescence of Prpl7 during spliceosome assembly to be
at an early assembly stage soon after the entry of U4/U6.U5 tri-snRNP and prior
to catalytic activation.
Demonstrated continued Prpl7 association with the spliceosome beyond the
completion of the splicing reactions.
Identified Avolp and Sgmlp as novel interacting partners of Prpl7 through a genome-wide two-hybrid screen.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/299 |
| Date | 07 1900 |
| Creators | Katoch, Aparna |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Rights | I grant Indian Institute of Science the right to archive and to make available my thesis or dissertation in whole or in part in all forms of media, now hereafter known. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. |
Page generated in 0.003 seconds