Cryptococcus neoformans Slu7 ensures nuclear positioning during mitotic progression through RNA splicing.

The position of the nucleus before it divides during mitosis is variable in different budding yeasts. Studies in the pathogenic intron-rich fungus Cryptococcus neoformans reveal that the nucleus moves entirely into the daughter bud before its division. Here, we report functions of a zinc finger motif containing spliceosome protein C. neoformans Slu7 (CnSlu7) in cell cycle progression. The budding yeast and fission yeast homologs of Slu7 have predominant roles for intron 3' splice site definition during pre-mRNA splicing. Using a conditional knockdown strategy, we show CnSlu7 is an essential factor for viability and is required for efficient cell cycle progression with major role during mitosis. Aberrant nuclear migration, including improper positioning of the nucleus as well as the spindle, were frequently observed in cells depleted of CnSlu7. However, cell cycle delays observed due to Slu7 depletion did not activate the Mad2-dependent spindle assembly checkpoint (SAC). Mining of the global transcriptome changes in the Slu7 knockdown strain identified downregulation of transcripts encoding several cell cycle regulators and cytoskeletal factors for nuclear migration, and the splicing of specific introns of these genes was CnSlu7 dependent. To test the importance of splicing activity of CnSlu7 on nuclear migration, we complemented Slu7 knockdown cells with an intron less PAC1 minigene and demonstrated that the nuclear migration defects were significantly rescued. These findings show that CnSlu7 regulates the functions of diverse cell cycle regulators and cytoskeletal components, ensuring timely cell cycle transitions and nuclear division during mitosis.

OsbZIP47 Is an Integrator for Meristem Regulators During Rice Plant Growth and Development.

Stem cell homeostasis by the WUSCHEL-CLAVATA (WUS-CLV) feedback loop is generally conserved across species; however, its links with other meristem regulators can be species-specific, rice being an example. We characterized the role of rice OsbZIP47 in vegetative and reproductive development. The knockdown (KD) transgenics showed meristem size abnormality and defects in developmental progression. The size of the shoot apical meristem (SAM) in 25-day OsbZIP47KD plants was increased as compared to the wild-type (WT). Inflorescence of KD plants showed reduced rachis length, number of primary branches, and spikelets. Florets had defects in the second and third whorl organs and increased organ number. OsbZIP47KD SAM and panicles had abnormal expression for CLAVATA peptide-like signaling genes, such as FON2-LIKE CLE PROTEIN1 (FCP1), FLORAL ORGAN NUMBER 2 (FON2), and hormone pathway genes, such as cytokinin (CK) ISOPENTEYLTRANSFERASE1 (OsIPT1), ISOPENTEYLTRANSFERASE 8 (OsIPT8), auxin biosynthesis OsYUCCA6, OsYUCCA7 and gibberellic acid (GA) biosynthesis genes, such as GRAIN NUMBER PER PANICLE1 (GNP1/OsGA20OX1) and SHORTENED BASAL INTERNODE (SBI/OsGA2ox4). The effects on ABBERANT PANICLE ORGANIZATION1 (APO1), OsMADS16, and DROOPING LEAF (DL) relate to the second and third whorl floret phenotypes in OsbZIP47KD. Protein interaction assays showed OsbZIP47 partnerships with RICE HOMEOBOX1 (OSH1), RICE FLORICULA/LEAFY (RFL), and OsMADS1 transcription factors. The meta-analysis of KD panicle transcriptomes in OsbZIP47KD, OsMADS1KD, and RFLKD transgenics, combined with global OSH1 binding sites divulge potential targets coregulated by OsbZIP47, OsMADS1, OSH1, and RFL. Further, we demonstrate that OsbZIP47 redox status affects its DNA binding affinity to a cis element in FCP1, a target locus. Taken together, we provide insights on OsbZIP47 roles in SAM development, inflorescence branching, and floret development.

Characterization of a new rice OsMADS1 null mutant generated by homologous recombination-mediated gene targeting.

MAIN CONCLUSION: A new, stable, null mutant of OsMADS1 generated by homologous recombination-based gene targeting in an indica rice confirms its regulatory role for floral meristem identity, its determinate development and floral organ differentiation. OsMADS1, an E-class MADS-box gene, is an important regulator of rice flower development. Studies of several partial loss-of-function and knockdown mutants show varied floret organ defects and degrees of meristem indeterminacy. The developmental consequences of a true null mutant on floret meristem identity, its determinate development and differentiation of grass-specific organs such as the lemma and palea remain unclear. In this study, we generated an OsMADS1 null mutant by homologous recombination-mediated gene targeting by inserting a selectable marker gene (hpt) in OsMADS1 and replacing parts of its cis-regulatory and coding sequences. A binary vector was constructed with diphtheria toxin A chain gene (DT-A) as a negative marker to eliminate random integrations and the hpt marker for positive selection of homologous recombination. Precise disruption of the endogenous OsMADS1 locus in the rice genome was confirmed by Southern hybridization. The homozygous osmads1ko null mutant displayed severe defects in all floral organs including the lemma and palea. We also noticed striking instances of floral reversion to inflorescence and vegetative states which has not been reported for other mutant alleles of OsMADS1 and further reinforces the role of OsMADS1 in controlling floral meristem determinacy. Our data suggest, OsMADS1 commits and maintains determinate floret development by regulating floral meristem termination, carpel and ovule differentiation genes (OsMADS58, OsMADS13) while its modulation of genes such as OsMADS15, OsIG1 and OsMADS32 could be relevant in the differentiation and development of palea. Further, our study provides an important perspective on developmental stage-dependent modulation of some OsMADS1 target genes.

Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features.

Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in Saccharomyces cerevisiae. However, Prp16 functions are largely unexplored in Schizosaccharomyces pombe, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5' splice site (5'SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5'SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the spprp16F528S mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5'SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in non-coding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in spprp16F528S cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site-snRNA interaction strength.

Regulation of meristem maintenance and organ identity during rice reproductive development.

Grasses have evolved complex inflorescences, where the primary unit is the specialized short branch called a spikelet. Detailed studies of the cumulative action of the genetic regulators that direct the progressive change in axillary meristem identity and their terminal differentiation are crucial to understanding the complexities of the inflorescence and the development of a determinate floret. Grass florets also pose interesting questions concerning the morphologies and functions of organs as compared to other monocots and eudicots. In this review, we summarize our current knowledge of the regulation of the transitions that occur in grass inflorescence meristems, and of the specification of floret meristems and their determinate development. We primarily use rice as a model, with appropriate comparisons to other crop models and to the extensively studied eudicot Arabidopsis. The role of MADS-domain transcription factors in floral organ patterning is well documented in many eudicots and in grasses. However, there is evidence to suggest that some of these rice floral regulators have evolved distinctive functions and that other grass species-specific factors and regulatory pathways occur - for example the LOFSEP 'E' class genes OsMADS1 and OsMAD34, and ramosa genes. A better understanding of these systems and the epigenetic regulators and hormone signaling pathways that interact with them will provide new insights into the rice inflorescence meristem and the differentiation of its floret organs, and should indicate genetic tools that can be used to control yield-related traits in both rice and other cereal crops.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

The Fission Yeast Pre-mRNA-processing Factor 18 (prp18+) Has Intron-specific Splicing Functions with Links to G1-S Cell Cycle Progression.

The fission yeast genome, which contains numerous short introns, is an apt model for studies on fungal splicing mechanisms and splicing by intron definition. Here we perform a domain analysis of the evolutionarily conserved Schizosaccharomyces pombe pre-mRNA-processing factor, SpPrp18. Our mutational and biophysical analyses of the C-terminal α-helical bundle reveal critical roles for the conserved region as well as helix five. We generate a novel conditional missense mutant, spprp18-5 To assess the role of SpPrp18, we performed global splicing analyses on cells depleted of prp18+ and the conditional spprp18-5 mutant, which show widespread but intron-specific defects. In the absence of functional SpPrp18, primer extension analyses on a tfIId+ intron 1-containing minitranscript show accumulated pre-mRNA, whereas the lariat intron-exon 2 splicing intermediate was undetectable. These phenotypes also occurred in cells lacking both SpPrp18 and SpDbr1 (lariat debranching enzyme), a genetic background suitable for detection of lariat RNAs. These data indicate a major precatalytic splicing arrest that is corroborated by the genetic interaction between spprp18-5 and spprp2-1, a mutant in the early acting U2AF59 protein. Interestingly, SpPrp18 depletion caused cell cycle arrest before S phase. The compromised splicing of transcripts coding for G1-S regulators, such as Res2, a transcription factor, and Skp1, a regulated proteolysis factor, are shown. The cumulative effects of SpPrp18-dependent intron splicing partly explain the G1 arrest upon the loss of SpPrp18. Our study using conditional depletion of spprp18+ and the spprp18-5 mutant uncovers an intron-specific splicing function and early spliceosomal interactions and suggests links with cell cycle progression.

Genome-Wide Targets Regulated by the OsMADS1 Transcription Factor Reveals Its DNA Recognition Properties.

OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5 This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Functions for rice RFL in vegetative axillary meristem specification and outgrowth.

Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport.

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice.

Polycomb Repressive Complex 2 (PRC2) represses the transcriptional activity of target genes through trimethylation of lysine 27 of histone H3. The functions of plant PRC2 have been chiefly described in Arabidopsis, but specific functions in other plant species, especially cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice results in complete sterility, and mutant flowers have severe floral organ defects and indeterminacy that resemble loss-of-function mutants in E-function floral organ specification genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and OsMADS34 as differentially expressed in the emf2b mutant compared with wild type. OsMADS1 and OsMADS6, known to be required for meristem determinacy in rice, have reduced expression in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by quantitative (q)RT-PCR showed that all three genes are presumptive targets of PRC2 in the meristem. Therefore, in rice, and possibly other cereals, PRC2 appears to play a major role in floral meristem determinacy through modulation of the expression of E-function genes.

Involvement of S. cerevisiae Rpb4 in subset of pathways related to transcription elongation.

Yeast Rpb4, a subunit of RNA pol II is not essential for viability but is involved in multiple cellular phenotypes such as temperature sensitivity, enhanced pseudohyphal morphology, and decreased sporulation. Both in vivo and in vitro studies strongly support involvement of Rpb4 in transcription initiation, while its role in transcription elongation is not entirely consistent. Here we show that Rpb4 is not required for recruitment of RNA pol II on the coding region of YLR454w, a representative long gene. Yet we find strong genetic interaction of rpb4∆ with mutants in many transcription elongation factors such as Paf1, Spt4, Dst1, Elp3 and Rpb9. We demonstrate that, Rpb4 interacts functionally with Paf1 to affect the transcription elongation of the FKS1 gene. Our results suggest that while Rpb4 is not required for general transcription elongation, it could support transcription elongation for specific of class of genes by interaction with other elongation factors.

Splicing functions and global dependency on fission yeast slu7 reveal diversity in spliceosome assembly.

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7(+) (spslu7(+)) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3' splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3' splice site distance, intron length, and the impact of its A/U content at the 5' end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1(+), a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways.

SEPALLATA (SEP) MADS box transcription factors mediate floral development in association with other regulators. Mutants in five rice (Oryza sativa) SEP genes suggest both redundant and unique functions in panicle branching and floret development. leafy hull sterile1/OsMADS1, from a grass-specific subgroup of LOFSEP genes, is required for specifying a single floret on the spikelet meristem and for floret organ development, but its downstream mechanisms are unknown. Here, key pathways and directly modulated targets of OsMADS1 were deduced from expression analysis after its knockdown and induction in developing florets and by studying its chromatin occupancy at downstream genes. The negative regulation of OsMADS34, another LOFSEP gene, and activation of OsMADS55, a SHORT VEGETATIVE PHASE-like floret meristem identity gene, show its role in facilitating the spikelet-to-floret meristem transition. Direct regulation of other transcription factor genes like OsHB4 (a class III homeodomain Leu zipper member), OsBLH1 (a BEL1-like homeodomain member), OsKANADI2, OsKANADI4, and OsETTIN2 show its role in meristem maintenance, determinacy, and lateral organ development. We found that the OsMADS1 targets OsETTIN1 and OsETTIN2 redundantly ensure carpel differentiation. The multiple effects of OsMADS1 in promoting auxin transport, signaling, and auxin-dependent expression and its direct repression of three cytokinin A-type response regulators show its role in balancing meristem growth, lateral organ differentiation, and determinacy. Overall, we show that OsMADS1 integrates transcriptional and signaling pathways to promote rice floret specification and development.

Plasmodium falciparum Prp16 homologue and its role in splicing.

Large numbers of Plasmodium genes have been predicted to have introns. However, little information exists on the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNA processing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, present in the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is a member of DEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experiments with the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis of ATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associated domain (HA2) reduced these activities considerably, indicating that the helicase-associated domain may regulate the PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N-terminal domain (1-80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasite cultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16p in splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16 protein, not the full length PfPrp16, can rescue the yeast prp16 temperature-sensitive mutant. These results suggest that although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtle differences exist with regards to its associated factors or its assembly with spliceosomes.

Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression.

The yeast Bud31 protein, a Prp19 complex (NTC) member, aids spliceosome assembly and thus promotes efficient pre-mRNA splicing. The bud31 null cells show mild budding abnormalities at optimal growth temperatures and, at higher temperatures, have growth defects with aberrant budding. Here we have assessed cell cycle transitions which require Bud31. We find Bud31 facilitates passage through G1-S regulatory point (Start) but is not needed for G2-M transition or for exit from mitosis. To co-relate Bud31 functions in cell division with splicing, we studied the splicing status of transcripts that encode proteins involved in budding. We find Bud31 promotes efficient splicing of only some of these pre-mRNAs, for example, ARP2 and SRC1. Wild type cells have a long and a short isoform of SRC1 mRNA and protein, out of which the shorter mRNA splice variant is predominant. bud31Δ cells show inefficient SRC1 splicing and entirely lack the shorter SRC1 spliced mRNA isoform. Yeast PRP17, another NTC sub-complex member, is also required for G1-S and G2-M cell cycle transitions. We examined genetic interactions between BUD31 and PRP17. While both factors were needed for efficient cell cycle dependent gene expression, our data indicate that distinct pre-mRNAs depend on each of these non-essential splicing factors.

Saccharomyces cerevisiae NineTeen complex (NTC)-associated factor Bud31/Ycr063w assembles on precatalytic spliceosomes and improves first and second step pre-mRNA splicing efficiency.

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a ∼25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.

Auxin-responsive OsMGH3, a common downstream target of OsMADS1 and OsMADS6, controls rice floret fertility.

GH3 proteins control auxin homeostasis by inactivating excess auxin as conjugates of amino acids and sugars and thereby controlling cellular bioactive auxin. Since auxin regulates many aspects of plant growth and development, regulated expression of these genes offers a mechanism to control various developmental processes. OsMGH3/OsGH3-8 is expressed abundantly in rice florets and is regulated by two related and redundant transcription factors, OsMADS1 and OsMADS6, but its contribution to flower development is not known. We functionally characterize OsMGH3 by overexpression and knock-down analysis and show a partial overlap in these phenotypes with that of mutants in OsMADS1 and OsMADS6. The overexpression of OsMGH3 during the vegetative phase affects the overall plant architecture, whereas its inflorescence-specific overexpression creates short panicles with reduced branching, resembling in part the effects of OsMADS1 overexpression. In contrast, the down-regulation of endogenous OsMGH3 caused phenotypes consistent with auxin overproduction or activated signaling, such as ectopic rooting from aerial nodes. Florets in OsMGH3 knock-down plants were affected in carpel development and pollen viability, both of which reduced fertility. Some of these floret phenotypes are similar to osmads6 mutants. Taken together, we provide evidence for the functional significance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development.

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis.

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.

Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture.

Activity of axillary meristems dictates the architecture of both vegetative and reproductive parts of a plant. In Arabidopsis thaliana, a model eudicot species, the transcription factor LFY confers a floral fate to new meristems arising from the periphery of the reproductive shoot apex. Diverse orthologous LFY genes regulate vegetative-to-reproductive phase transition when expressed in Arabidopsis, a property not shared by RFL, the homolog in the agronomically important grass, rice. We have characterized RFL by knockdown of its expression and by its ectopic overexpression in transgenic rice. We find that reduction in RFL expression causes a dramatic delay in transition to flowering, with the extreme phenotype being no flowering. Conversely, RFL overexpression triggers precocious flowering. In these transgenics, the expression levels of known flowering time genes reveal RFL as a regulator of OsSOC1 (OsMADS50), an activator of flowering. Aside from facilitating a transition of the main growth axis to an inflorescence meristem, RFL expression status affects vegetative axillary meristems and therefore regulates tillering. The unique spatially and temporally regulated RFL expression during the development of vegetative axillary bud (tiller) primordia and inflorescence branch primordia is therefore required to produce tillers and panicle branches, respectively. Our data provide mechanistic insights into a unique role for RFL in determining the typical rice plant architecture by regulating distinct downstream pathways. These results offer a means to alter rice flowering time and plant architecture by manipulating RFL-mediated pathways.