Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side. Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules. By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.

At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.

No description available.

No description available.

Gene deletions are a powerful method to uncover the cellular functions of a given gene in living systems. A limitation to this methodology is that it is not applicable to essential genes. Even for non-essential genes, gene knockouts cause complete absence of gene product thereby limiting genetic analysis of the biological pathway. Alternatives to gene deletions are mutants that are conditional, for e.g, temperature sensitive (ts) mutants are robust tools to understand temporal and spatial functions of genes. By definition, products of such mutants have near normal activity at a lower temperature or near-optimal growth temperature which is called as the permissive temperature and reduced activity at a higher, non-optimal temperature called as the non-permissive temperature. Generation of ts alleles in genes of interest is often time consuming as it requires screening a large population of mutants to identify those that are conditional. Often many essential proteins do not yield ts such alleles even after saturation mutagenesis and extensive screening (Harris et al., 1992; Varadarajan et al., 1996). The limited availability of such mutants in many essential genes prompted us to adopt a biophysical approach to design temperature-sensitive missense mutants in an essential gene of fission yeast. Several studies report that mutations in buried or solvent-inaccessible amino acids cause extensive changes in the thermal stability of proteins and specific substitutions create temperature-sensitive mutants (Rennell et al., 1991; Sandberg et al., 1995). We used the above approach to generate conditional mutants in the fission yeast gene spprp18+encoding an essential predicted second splicing factor based on its homology with human and S. cerevisiae proteins. We have used a missense mutant coupled with a conditional expression system to elucidate the cellular functions of spprp18+. Further, we have employed the same biophysical principle to generate a missense mutant in spago1+ RNA silencing factor that is non-essential for viability but has critical functions in the RNAi pathway of fission yeast. Fission yeast pre-mRNA splicing: cellular functions for the protein factor SpPrp18 Pre-mRNA splicing is an evolutionarily conserved process that excises introns from nascent transcripts. Splicing reactions are catalyzed by the large ribonuclear protein machinery called the spliceosome and occur by two invariant trans-esterification reactions (reviewed in Ruby and Abelson, 1991; Moore et al., 1993). The RNA-RNA, RNA–protein and protein-protein interactions in an assembly of such a large protein complex are numerous and highly dynamic in nature. These interactions in in vitro splicing reactions show ordered recruitment of essential small nuclear ribonucleic particles snRNPs and non–snRNP components on pre-mRNA cis-elements. Further these trans acting factors recognize and poise the catalytic sites in proximity to identify and excise introns. The precision of the process is remarkable given the diversity in architecture for exons and introns in eukaryotic genes (reviewed in Burge et al., 1999; Will and Luhrmann, 2006). Many spliceosomal protein components are conserved across various organisms, yet introns have diverse features with large variations in primary sequence. We hypothesize that co-evolution of splicing factor functions occurs with changes in gene and intron architectures and argue for alternative spliceosomal interactions for spliceosomal proteins that thus enabling splicing of the divergent introns. In vitro biochemical and genetic studies in S. cerevisiae and biochemical studies with human cell lines have indicated that ScPRP18 and its human homolog hPRP18 function during the second catalytic reaction. In S. cerevisiae, ScPrp18 is non-essential for viability at growth temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Horowitz and Abelson, 1993b). The concerted action of ScSlu7 - ScPrp18 heteromeric complex is essential for proper 3’ss definition during the second catalytic reaction (Zhang and Schwer, 1997; James et al., 2002). These in vitro studies also hinted at a possible intron -specific requirement for ScPrp18 and ScSlu7 factors as they were dispensable for splicing of intron variants made in modified ACT1 intron containing transcripts (Brys and Schwer, 1996; Zhang and Schwer, 1997). A short spacing distance between branch point adenosine to 3’splice site rendered the substrate independent of Prp18 and Slu7 for the second step (Brys and Schwer, 1996; Zhang and Schwer, 1997). Extensive mutational analyses of budding yeast ScPrp18 identified two functional domains and suggested separate roles during splicing (Bacikova and Horowitz, 2002; James et al., 2002). Fission yeast with its genome harboring multiple introns and degenerate splice signals has recently emerged as a unique model to study relationships between splicing factors and their role in genomes with short introns. Previously, studies in our lab had initiated genetic and mutational analysis of S. pombe Prp18, the predicted homolog of budding yeast Prp18. Genetic analysis showed its essentiality, but a set of missense mutants based on studies of budding yeast ScPrp18 (Bacikova and Horowitz, 2002) gave either inactive null or entirely wild type phenotype for the fission yeast protein. In this study, we have extended our previous mutational analysis of fission yeast Prp18 by adopting biophysical and computational approaches to generate temperature-sensitive mutants. A missense mutant was used to understand the splicing functions and interactions of SpPrp18 and the findings are summarized below. Fission yeast SpPrp18 is an essential splicing factor with transcript-specific functions and links efficient splicing with cell cycle progression We initiated our analysis of SpPrp18 by adopting a biophysical approach to generate ts mutants. We used the PREDBUR algorithm to predict a set of buried residues, which when mutated could result in a temperature-sensitive phenotype that complements the null allele at permissive temperature. These predictions are based upon two biophysical properties of amino acids: 1) Hydrophobicity, which is calculated in a window of seven amino acids 2) Hydrophobic moment, which is calculated in a sliding window of nine amino acids in a given protein sequence. Several studies correlate these properties to protein stability and function (Varadarajan et al., 1996). One of the buried residue mutants V194R, in helix 1 of SpPrp18 conferred weak temperature- sensitivity and strong cold-sensitivity even when the protein was over expressed from a plasmid. Through semi-quantitative RT-PCR we showed splicing-defects for tfIId+ intron1 in these cells even when grown at permissive temperature. The primary phenotype was the accumulation of pre-mRNA. Further, we showed this splicing arrest is co-related with reduced levels of SpPrp18 protein, linking protein stability and splicing function. Next we examined the effects of this mutation on function by further reduction of protein levels. This was done by integrating the expression cassette nmt81:spprp18+/spprp18V194R at the leu1 chromosomal locus and by metabolic depletion of the integrated allele. Through RT-PCRs we demonstrated that depletion of wild type or missense protein has intron specific splicing defects. These findings showed its non-global and possibly substrate-specific splicing function. In the affected introns, precursor accumulation is the major phenotype, confirming prior data from our lab that hinted at its likely early splicing role. This contrasts with the second step splicing role of the human or budding yeast Prp18 proteins. Previous data from our lab showed loss of physical interaction between SpPrp18 and SpSlu7 by co-immunoprecipitation studies. This again differs from the strong and functionally important ScPrp18 and ScSlu7 interaction seen in budding yeast. We show the absence of charged residues in SpSlu7 interaction region formed by SpPrp18 helix1 and helix2 which can explain the altered associations for SpPrp18 in fission yeast. Importantly, as the V194R mutation in helix 1 shows splicing defects even at permissive temperature, the data indicate a critical role for helix 1 for splicing interactions, possibly one that bridges or stabilizes the proposed weak association of SpPrp18-SpSlu7 with a yet unknown splicing factor. We also investigated the effects of mutations in other helices; surprisingly we recovered only mutations with very subtle growth phenotypes and very mild splicing defects. Not surprisingly, stop codon at L239 residue predicted to form a truncated protein lacking helices 3, 4 and 5 conferred recessive but null phenotype implicating essential functions for other helices. Other amino acid substitutions at L239 position had near wild type phenotype at 30°C and 37°C. Helix 3 buried residue mutant I259A conferred strong cold-sensitivity when over expressed from plasmid, but semi quantitative analysis indicated no splicing defects for intron1 in the constitutively expressed transcript tfIId+. These findings indicate cold sensitivity either arises due to compromised splicing of yet unknown transcripts or that over-expressed protein has near wild type activity. We find mutations in the helix 5 buried residues L324 also conferred near WT phenotype. Earlier studies in the lab found that substitution of surface residues KR that are in helix 5 with alanine lead to null phenotypes (Piyush Khandelia and Usha Vijayraghavan unpublished data). We report stable expression of all of these mutant proteins; L239A, L239P, L239G, I259A, I259V, L324F, L324A as determined by our immunoblot analysis at 30°C and 37°C. The mild phenotypes of many buried residues can be attributed to orientation of their functional groups into a protein cavity between the helices. Lastly, our microscopic cellular and biochemical analysis of cellular phenotypes of spprp18 mutant provided a novel and direct role of this factor in G1-S transition of cell cycle. Our RT-PCR data suggest spprp18+ is required for efficient splicing of several intron containing transcripts involved in G1-S transition and subsequent activation of MBF complex (MluI cell cycle box-binding factor complex) during S-phase and shows a mechanistic link between cell cycle progression and splicing. A tool to study links between RNA interference, centromeric non-coding RNA transcription and heterochromatin formation S.pombe possesses fully functional RNA interference machinery with a single copy for essential RNAi genes ago1+, dcr1+ and rdp1+. Deletion of any of these genes causes loss of heterochromatinzation with abnormal cytokinesis, cell-cycle deregulation and mating defects (Volpe et al., 2002). In S.pombe, exogenous or endogenously generated dsRNA’s from transcription of centromeric repeats are processed by the RNaseIII enzyme dicer to form siRNA. These siRNA’s are loaded in Ago1 to form minimal RNA induced silencing complex (RISC) complex or specialized transcription machinery complex RNA induced transcriptional silencing (RITS) complex and target chromatin or complementary mRNAs for silencing. Thus as in other eukaryotes, fission yeast cells deploy RNAi mediated silencing machinery to regulate gene-expression and influence chromatin status. Several recent studies point to emerging new roles of RNAi and its association with other RNA processes (Woolcock et al., 2011; Bayane et al., 2008; Kallgren et al., 2014). Many recent reports suggest physical interactions of RISC or RITS and RNA dependent RNA polymerase complex (RDRC) with either some factors of the spliceosomal machinery, heterochromatin machinery (CLRC complex) and the exosome mediated RNA degradation machinery (Bayne et al., 2008 and Chinen et al., 2010 ; Hiriart et al., 2012; Buhler et al., 2008; Bayne et al., 2010 ). Thus we presume conditional alleles in spago1+ will facilitate future studies to probe the genetic network between these complexes as most analyses thus far rely on ago1∆ allele or have been based on proteomic pull down analyses of RISC or RITS complexes. In this study, we employed biophysical and modeling approaches described earlier to generate temperature sensitive mutants in spago1+ and spdcr1+. We tested several mutants for their ability to repress two reporter genes in a conditional manner. Our modeling studies on SpAgo1 PAZ domain indicated structural similarities with human Ago1 PAZ domain. We created site-directed missense mutants at predicted buried residues or in catalytic residues. We also analyzed the effects of random amino acid replacements in specific predicted buried or catalytic residues of SpAgoI. These ago1 mutants were screened as pools for their effects on silencing of GFP or of ura4+ reporter genes. These assays assessed post transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) activity of these mutants. We obtained three temperature sensitive SpAgo1 mutants V324G, V324S and L215V while the V324E replacement was a null allele. Based upon our modeling, a likely explanation for the phenotype of these mutants is structural distortion or mis-orientation of the functional groups caused due to these mutations, which affect activity in a temperature dependent manner. This distortion in the PAZ domain may affect binding of siRNA and thereby lead to heterochromatin formation defects that we observed. Our data on the SpAgo1 V324 mutant shows conditional centromeric heterochromatin formation confirmed by semi quantitative RT-PCR for dh transcripts levels that shows temperature dependent increase in these transcripts. We find reduced H3K9Me2 levels at dh locus by chromatin immunoprecipitation (ChIP) assay, linking the association of siRNAs for establishment of heterochromatin at this loci. The data on PTGS of GFP transcripts show SpAgo1 V324G mutation has decreased slicing activity as semi-quantitative RT-PCR for GFP transcripts show increased levels at non permissive temperature. These studies point out the importance of siRNA binding to the PAZ domain and its effect on slicing activity of SpAgo1. The mutations in Y292 showed residue loss of centromeric heterochromatin formation phenotype. Thus, we ascribe critical siRNA binding and 3’ end recognition functions to this residue of SpAgo1. These studies point out functional and structural conservation across hAgo1 and SpAgo1. Adopting the aforementioned biophysical mutational approach, we generated mutants in spdcr1+ and screened for those with conditional activity. Our modeling studies on SpDcr1 helicase domain shows it adopts the conserved helicase domain structure seen for other DEAD Box helicases. Our data on mutational analysis of a conserved buried residue I143 in the walker motif B created inactive protein. The data confirm critical functions for dicer in generation of siRNAs and also in recognition of dsRNA ends. Mutants in buried residues L1130 and I1228 of RNase IIIb domain were inactive and the proximity of these residues to the catalytic core suggest that the critical structural alignment of catalytic residues is indispensable for carrying out dsRNA cleavage to generate siRNAs. We also attribute critical catalytic functions to SpDcr1 D1185 residue for generation of siRNA and heterochromatin formation as measured by our transcriptional gene silencing assay. Our studies employing biophysical and computational approaches to design temperature-sensitive mutants have been successfully applied to an essential splicing factor SpPrp18, which was refractory for ts mutants by other methods. Using a missense mutant, we showed its intron-specific splicing function for subsets of transcripts and deduced that its ubiquitous splicing role is arguable. We have uncovered a link between the splicing substrates of SpPrp18 and direct evidence of splicing based cell cycle regulation, thus providing a mechanistic link to the cell cycle arrest seen in some splicing factor mutants. The same methodology was applied to another important biological pathway, the RNAi machinery, where central factors SpAgoI and SpDcrI were examined We report the first instance of conditional gene silencing tool by designing Ago1 ts mutants which will be useful for future studies of the global interaction network between RNAi and other RNA processing events.

Nuclear pre-mRNA splicing proceeds via two mechanistically conserved consecutive trans-esterification reactions catalyzed by the spliceosome. The ordered coalescence of spliceosomal snRNPs and splicing factors on the pre-mRNA, coupled with essential spliceosomal rearrangements poise the splice-sites in proximity for the two catalytic reactions, ensuring intron removal and exon ligation to yield functional mRNA (reviewed in Will and Lührmann, 2006). Scope of the study The S. cerevisiae splicing factors Prp18 and Slu7 and their human homologs function during second catalytic reaction. In S. cerevisiae, Slu7 is essential, whereas Prp18 is dispensable at temperatures <30°C (Vijayraghavan et al., 1989; Vijayraghavan and Abelson, 1990; Frank et al., 1992; Horowitz and Abelson, 1993b; reviewed in Umen and Guthrie, 1995). Slu7 acts in concert with Prp18 and their direct interaction is required for their stable spliceosomal association (Zhang and Schwer, 1997; James et al., 2002). In vitro studies indicate that both the factors are dispensable for splicing of introns with short distances between branch nucleotide to 3’ splice-site (Brys and Schwer, 1996; Zhang and Schwer, 1997). Furthermore, mutational analyses of Slu7 and Prp18 have defined their functional domains/motifs (Frank and Guthrie, 1992; Bacíková and Horowitz, 2002; James et al., 2002). In this study, we have examined functions for the predicted homologs of Slu7 and Prp18 in fission yeast; an evolutionarily divergent organism where splicing mechanisms are not well understood and whose genome harbors genes with predominantly multiple introns with degenerate splice-junction sequences. Towards this goal, a combinatorial approach employing genetic and biochemical methods was undertaken to understand splicing functions and interactions of SpSlu7 and SpPrp18. Our mutational analysis of these protein factors provided an overview of the domains/motifs critical for their in vivo functions. Lastly our analysis of components of the budding yeast Cef1p-associated complex show novel interactions and splicing functions for two uncharacterized, yet evolutionarily conserved proteins. Conserved fission yeast splicing proteins SpSlu7 and SpPrp18 are essential for pre-mRNA splicing but have altered spliceosomal associations and functions Analyzing conserved splicing factors in evolutionarily divergent organisms is an important means to gain deeper functional insights on splicing mechanisms in genomes with varied gene architecture. We initiated our analysis of the ‘predicted’ S. pombe second-step splicing factors: spprp18+ and spslu7+, by genetically depleting these factors. We find spprp18+ is essential for viability, unlike budding yeast PRP18; while SLU7 is essential in both yeasts. The complete essentiality of both these fission yeast factors, prompted us to create conditional-lethal thiamine repressible ‘switch-off’ strains to probe their splicing functions. Through semi-quantitative RT-PCR and northern blot analysis we demonstrate splicing defects for tfIId+ pre-mRNAs upon metabolic depletion of spprp18+ or spslu7+, thus linking their essentiality to a role in pre-mRNA splicing. Further we examined whether their requirement as splicing factors is governed by specific intronic features. We find both factors are required in vivo for removal of several introns. However, for the introns tested, their functions are not strictly correlated with intron length, number, position or the branch-nucleotide to 3’ splice-site distance. The latter features dictate the need for their S. cerevisiae homologs. Strikingly the lack of either one of these essential proteins, arrests splicing before the first catalytic step; implicating possible functions early in spliceosome assembly even before any catalytic event, as opposed to budding yeast Slu7 and Prp18, which are second-step factors assembling late onto the spliceosome after the first splicing reaction. Given the different splicing arrest point, on depletion of SpSlu7 and SpPrp18, we investigated through yeast two-hybrid and co-immunoprecipitation assays whether the direct interaction between these proteins is conserved. We find despite being nuclear localized these proteins do not interact in either of the assays employed. A structural basis for the lack of interaction was provided by our homology modeling of SpPrp18, that was based on the crystal structure of S. cerevisiae Prp1879 (Jiang et al., 2000). Together these data raise the possibility of contextual functions and interactions for these conserved proteins that varies with changes in gene architecture. This likelihood is strengthened by our reciprocal genetic complementation tests; wherein we find that SpSlu7 and SpPrp18 cannot complement the corresponding S. cerevisiae null alleles and vice versa. Additionally, the human homologs, hSlu7 and hPrp18 also failed to rescue null alleles for spslu7+ and spprp18+. To understand the likely point of coalescence of SpSlu7 and SpPrp18 on assembling spliceosomes, we probed their snRNP associations through co-immunoprecipitation analysis. Our data revealed interaction of SpSlu7 with the U2, U5 and U6 snRNPs at moderate salt concentrations with the interaction with U5 snRNP being retained at higher salt conditions. SpPrp18, on the other hand, showed only a very weak association with U5 snRNP. Our analysis thus indicates that the assembly and step of action for “predicted” late-acting splicing factors in fission yeast differs from that in budding yeast, implicating novel interactions and functions for these fission yeast splicing factors. Mutational analysis of fission yeast SpPrp18 and SpSlu7 identifies functional domains To examine the protein domains/motifs critical for the functions of SpPrp18 and SpSlu7, we have performed a mutational study. This analysis was important after our findings that these factors are early acting and do not interact. The data gathered would shed light on the contribution of different domains/motifs in the functional diversification of these factors. Guided by the findings of Bacíková and Horowitz (2002); site-specific missense mutants were created in the highly conserved carboxyl-terminus (CR domain and helix 5) of SpPrp18. Additionally, site-specific missense mutants were generated in a conserved amino-terminus domain that is absent in budding yeast Prp18. Our data showed mutants in the highly conserved helix 5 and the CR domain of SpPrp18 to be recessive and non-functional, despite being stably expressed. This contrasts with the temperature-sensitivity conferred by similar mutants in homologous residues in budding yeast Prp18 (Bacíková and Horowitz, 2002). We speculate that the essentiality of the CR domain and helix 5 mutants of SpPrp18 arises due to a defect in spliceosomal association. However, the mutants in conserved residues in the protein’s amino-terminal domain are phenotypically wild type at various growth temperatures tested, suggesting redundant functions for these residues. Our data, based on analysis of a single missense mutant in the highly conserved zinc knuckle motif of SpSlu7, ascribes essential functions for the zinc knuckle motif. We find the mutant to be recessive and non-functional despite stable expression and normal cellular localization of the mutant protein. This contrasts with the behavior of zinc knuckle mutants in budding yeast and human Slu7. Budding yeast Slu7 mutants are functionally wild type and human Slu7 mutants have an altered cellular localization (Frank and Guthrie, 1992; James et al., 2002; Shomron et al., 2004). Possible roles for the zinc knuckle motif of SpSlu7 could be in facilitating interaction of SpSlu7 with U5 snRNA or even with some protein factor. Functional analysis of budding yeast Cef1p-associated complex SpSlu7 and its budding yeast homolog ScSlu7 co-purify with Cdc5/Cef1 in a complex of ~30 proteins together with U2, U5 and U6 snRNAs (Gavin et al., 2002; Ohi et al., 2002). Functional characterization of six proteins of the budding yeast Cef1p complex: Ydl209c (Cwc2/Ntc40), Ycr063w (Cwc14/Bud31), Yju2 (Cwc16), Ygr278w (Cwc22), Ylr424w (Spp382/Ntr1) and Ygl128c (Cwc23) was initiated using a combination of genetic and biochemical approaches. We probed direct protein-protein interactions between members of the Cef1p-associated complex by yeast two-hybrid assays. We also examined the pre-mRNA splicing roles for an essential factor, Yju2/Cwc16 and for a non-essential factor, Ycr063w/Cwc14. Our data reveals direct interaction between Yju2 and early acting factors, Syf1/Ntc90 and Clf1/Ntc77. Similarly interaction of Ydl209c/Cwc2 with early acting splicing factors, Prp19, Syf1/Ntc90 and Clf1/Ntc77 was noted. We created a temperature-sensitive expression strain for YJU2 using a temperature-sensitive Gal4 transcription trans-activator (Chakshusmathi et al., 2004; Mondal et al., 2007) to interrogate the splicing functions of YJU2. RT-PCR and northern blot assays show that depletion of YJU2 causes splicing defects for intron containing pre-mRNAs. We predict early splicing functions for YJU2 as is known for its interacting partners. Furthermore, we find that genetic depletion of the non-essential factor YCR063w causes temperature-sensitivity as has been reported for a few other factors (for e.g. Prp17, Lea1, Snt309/Ntc25, Ecm2) of Cef1p-associated complex (Jones et al, 1995; Chen et al., 1998). Although our yeast two-hybrid data does not reveal any direct interactions between Ycr063w and other proteins of the Cef1p-associated complex, we probed its functions through in vitro splicing assays. Splicing extracts from ycr063w/ycr063w cells show compromised second-step splicing at higher temperatures, thereby implying an auxiliary function for Ycr063w in stabilizing some functionally critical interactions during splicing. These studies employing complementary genetic and biochemical approaches implicate functional divergence of conserved predicted ‘second-step’ fission yeast factors, SpSlu7 and SpPrp18, suggesting co-evolution of splicing factors with changes in genome architecture and intron-exon structure. Our studies on Cef1p-associated complex show novel interactions and implicate pre-mRNA splicing functions for two previously uncharacterized proteins.

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Comment les cellules établissent leurs formes et organisations internes est un problème biologique fondamental. Au cours de cette thèse, j’ai étudié le développement de la forme cellulaire et de la polarité chez la cellule de levure fissipare. Ces études sont fondées sur l’exploration de la façon dont les petites spores symétriques de levures se développent et s’organisent pour briser la symétrie pour la définition de leur tout premier axe de polarité. Dans une première partie, j’ai étudié les couplages entre la mécanique de surface de la paroi cellulaire des spores et la stabilité de domaines de polarité de Cdc42 qui contrôlent les aspects spatio-temporelles de la brisure de symétrie de ces spores. Dans une seconde partie, j’ai étudié les mécanismes par lesquels ces domaines de polarité contrôlent leur taille et l'adapte à la géométrie de la cellule, un processus vraisemblablement pertinents pour comprendre comment des domaines fonctionnels corticaux s’adaptent à la taille des cellules. Globalement, ces nouvelles recherches focalisant sur la façon dont les cellules développent dynamiquement leur forme et polarité de novo, permettent de mettre en évidence des couplages complexes dans la morphogenèse qui ne peuvent pas être testés en regardant les cellules à « l’état stationnaire» ou avec des outils génétiques. / How cells establish their proper shapes and organization is a fundamental biological problem. In this thesis, I investigated the dynamic development of cellular form and polarity in the rod-shape fission yeast cell. These studies are based on monitoring how small symmetric fission yeast spores grow and self-organize to break symmetry for the definition of their very first polarity axis. In a first part, I studied interplays between surface mechanics of the spore cell wall and the stability of Cdc42-based polarity domains which control spatio-temporal aspects of spore symmetry breaking. In a second part, I studied mechanisms by which these polarity domains control their width and adapt it to cell surface geometry, a process likely relevant to understand how functional cortical domains scale to cell size. Overall these novel investigations focusing on how cells dynamically develop their form and polarity de novo highlight complex feedbacks in morphogenesis that cannot be evidenced by looking at cells at “steady state” or with genetics.