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Usha Vijayraghavan
Ph. D. (California Institute of Technology, USA)
Phone: +91 80 22932681 / +91 80 23600168
E-mail: uvr@mcbl.iisc.ernet.in

Our interests are to understand eukaryotic gene regulation at the transcriptional and post-transcriptional levels using molecular genetics and functional genomics.  The effects of such regulated gene expression on cell division and differentiation are being studied.
Molecular genetic studies of pre-mRNA splicing factors:  Removal of introns from precursor-mRNAs occurs in a Ribonucleoprotein particle: the spliceosome whose assembly is necessary for the two pre-mRNA splicing reactions.  We use genetic and biochemical approaches to study spliceosome assembly, splicing reactions and the impact of pre-mRNA splicing on global gene expression.

The functions, interactions and partners of Prp17, Prp18 and Slu7 are being studied in budding yeast (Sc) and fission yeast (Sp).  In budding yeast all three proteins play a role in 3’splice-site definition during the second reaction.  We are investigating whether their functions are ubiquitous or are context dependent.  While ScPrp17 facilitates second-step splicing it is non-essential for growth and for in vitro splicing at low temperatures.  By using splicing-sensitive DNA microarrays and prp17 mutants the consequences on splicing and global transcript levels was examined.  This was done to probe if cellular pre-mRNAs dependent on Prp17 share any specific intronic features.  We find budding yeast introns longer than 200 nucleotides are Prp17-dependent for their splicing.  We are also examining functions for fission yeast homologs of PRP17, PRP18 and SLU7 as this organism has predominantly short introns with only moderately conserved consensus elements.  Consistent with our predictions based on budding yeast Prp17 we see that its fission yeast homolog- SpPrp17, is entirely non-essential for growth at all temperatures.  This correlates with the pombe genome having largely short introns.  These data also suggest that Prp17 functions are perhaps more relevant for splicing of long introns in other genomes.  On the other hand our investigations of two other splicing factors, from these two microbial models, suggest that while these factors are evolutionarily conserved their spliceosome associations and interactions vary.  In budding yeast ScPrp18 is non-essential but its fission yeast homolog SpPrp18 is entirely essential.  Slu7 is essential in both yeasts.  Repression of expression of pombe SpSlu7 or SpPrp18 causes splicing defects for a number of cellular pre-mRNAs including those with very short branch nucleotide to 3’splice site distances (~9nts).  Interestingly, emerging results point to an unexpected early splicing role for both these pombe proteins unlike the second-step function seen from in vitro analyses of their budding yeast or human homologs.  Thus our findings indicate divergence in the interactions and possibly functions for these pombe splicing factors.  Our current efforts aim at a mutational analysis of these fission proteins to decipher their structure-function relationship, interactions with partners and their global requirement for splicing and gene expression.

Regulatory genes controlling cell fate and cell proliferation during flower development :  Our overall goals are to understand the network of interactions between transcription factors and signaling molecules that controls organ formation from meristems (with stem cells).  As a model system we study the formation of the rice flowering stem (inflorescence) and floral organs to elucidate functions for transcription factors in patterning.RFL- the rice homologue of LFY, a key transcription activator of floral differentiation in Arabidopsis, is expressed in a profile distinct from that of many its plant homologues.  The regulated profile of RFL expression; high-level expression in the apical meristem coincident with a switch from vegetative to reproductive growth, its dynamic expression in the branching inflorescence meristem much in advance of floret formation, is known for a decade now.  Our studies have delineated novel cis-acting transcription enhancers elements located in RFL intron 1 and 2 confer this distinct expression pattern of this predicted transcription factor.  The functional importance of this regulated expression was examined by employing RNA interference to knockdown RFL expression in transgenic plants.  In complementary analyses we probed the effects of its overexpression.  All together our studies showed, for the first time, that RFL regulates two important features of the rice plant, i.e., flowering time and branching which is an important feature governing plant morphology.  These analyses also illustrated how novel functions can arise for evolutionarily conserved transcription factors by alterations in their expression domains.  We are now engaged in determining RFL regulated target genes by global transcript analysis of knockdown plants.  The indications are that RFL controls the expression of other transcription factors or factors in hormone signaling pathways.  Such functional genomics analyses will be exploited to decipher the mechanism of action of this regulator of plant form.  Another rice transcription factor under investigation is OsMADS1, a gene expressed in floral meristems and first (lemma and palea) and fourth whorl (carpel) floral organs.  OsMADS1 knockdown perturbs the differentiation of specific cell types in the lemma and palea; conversely, ectopic OsMADS1 expression suffices to direct lemma-like differentiation in the glume.  Our data implicate a direct role for OsMADS1 in patterning the rice first whorl floral organs and a regulatory role for the development of the inner floral organs.  Here too using a combination of transcript profiling of knockdown florets, global expression analysis on induction of OsMADS1, and by using DNA-protein interaction studies we aim to determine its mechanism of action as a regulator of floral organ differentiation.

Selected Publications

Nandi, A. K., Kushalappa, M. K., Prasad, K. and Vijayraghavan, U. (2000) Conserved function for the Arabidopsis SUPERMAN gene in establishing floral whorl boundaries in rice a monocotyledonous plant. Current Biology 10:215-218

Chawla, G., Sapra, A.K., Surana, U. and Vijayraghavan, U. (2003). Dependence of  Pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions.  Nucleic Acids Research 31:2333-2343

Sapra A.K., Arova, Y., Khandelia, P. and Vijayraghavan, U. (2004).  Genome-wide analysis of pre-mRNA splicing: intron features govern the requirement for the second-step factor, Prp17, in Saccharomyces cerevisae and Schizosaccaromyces pombe.  Journal of Biological Chemistry 279: 52437-52446 (Paper of the week, Editors choice)

Prasad, K., Sriram, P., and Vijayraghavan , U. (2005). OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs. The Plant Journal 43: 915-923

Yadav, S. R., Prasad, K., and Vijayraghavan, U.  (2007).  Divergent regulatory OsMADS2 functions control size, shape and differentiation of the highly derived rice floret second-whorl organ. Genetics 176:283-294.
Rao, N., Prasad, K., Ravikumar, P., and Vijayraghavan, U.  (2008).  Distinct regulatory role for RFL, the rice LFY homolog, in determining flowering time and plant architecture.  Proc. Natl. Acad. Sci., U.S.A. 105:3646-3651.