Saiprasad G. Palusa

Regulation of Plant Developmental Processes by a Novel Splicing Factor

Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

Extensive coupling of alternative splicing of pre‐mRNAs of serine/arginine (SR) genes with nonsense‐mediated decay

In Arabidopsis, pre-mRNAs encoding serine/arginine (SR) proteins, key regulators of constitutive and alternative splicing, are extensively alternatively spliced. In seedlings, 13 SR genes are alternatively spliced to generate 75 transcripts, of which 53 contain a premature termination codon (PTC). However, it is not known if any of the PTC-containing splice variants are the targets of nonsense-mediated decay (NMD) and if there is any link between NMD and the abundance of functional transcripts. Here, we analyzed the abundances of all splice variants for each alternatively spliced gene in an Arabidopsis mutant that lacks UPF3, one of the core components of NMD machinery, to determine if the PTC-containing transcripts are degraded by NMD. Our results show that about half of the 53 splice variants with a PTC are the targets of degradation by NMD. The accumulation of PTC-containing transcripts resulted in concomitant reduction in the amount of functional transcript. These results show widespread coupling of alternative splicing with NMD in the SR gene family, suggesting a strong link between unproductive splicing and the abundance of functional transcripts.

Picornavirus Modification of a Host mRNA Decay Protein

ABSTRACT Due to the limited coding capacity of picornavirus genomic RNAs, host RNA binding proteins play essential roles during viral translation and RNA replication. Here we describe experiments suggesting that AUF1, a host RNA binding protein involved in mRNA decay, plays a role in the infectious cycle of picornaviruses such as poliovirus and human rhinovirus. We observed cleavage of AUF1 during poliovirus or human rhinovirus infection, as well as interaction of this protein with the 5′ noncoding regions of these viral genomes. Additionally, the picornavirus proteinase 3CD, encoded by poliovirus or human rhinovirus genomic RNAs, was shown to cleave all four isoforms of recombinant AUF1 at a specific N-terminal site in vitro. Finally, endogenous AUF1 was found to relocalize from the nucleus to the cytoplasm in poliovirus-infected HeLa cells to sites adjacent to (but distinct from) putative viral RNA replication complexes. IMPORTANCE This study derives its significance from reporting how picornaviruses like poliovirus and human rhinovirus proteolytically cleave a key player (AUF1) in host mRNA decay pathways during viral infection. Beyond cleavage of AUF1 by the major viral proteinase encoded in picornavirus genomes, infection by poliovirus results in the relocalization of this host cell RNA binding protein from the nucleus to the cytoplasm. The alteration of both the physical state of AUF1 and its cellular location illuminates how small RNA viruses manipulate the activities of host cell RNA binding proteins to ensure a faithful intracellular replication cycle. This study derives its significance from reporting how picornaviruses like poliovirus and human rhinovirus proteolytically cleave a key player (AUF1) in host mRNA decay pathways during viral infection. Beyond cleavage of AUF1 by the major viral proteinase encoded in picornavirus genomes, infection by poliovirus results in the relocalization of this host cell RNA binding protein from the nucleus to the cytoplasm. The alteration of both the physical state of AUF1 and its cellular location illuminates how small RNA viruses manipulate the activities of host cell RNA binding proteins to ensure a faithful intracellular replication cycle.

Identification of an intronic splicing regulatory element involved in auto-regulation of alternative splicing of SCL33 pre-mRNA

In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.

Alternative splicing at NAGNAG acceptors in Arabidopsis thaliana SR and SR-related protein-coding genes

BackgroundSeveral recent studies indicate that alternative splicing in Arabidopsis and other plants is a common mechanism for post-transcriptional modulation of gene expression. However, few analyses have been done so far to elucidate the functional relevance of alternative splicing in higher plants. Representing a frequent and universal subtle alternative splicing event among eukaryotes, alternative splicing at NAGNAG acceptors contributes to transcriptome diversity and therefore, proteome plasticity. Alternatively spliced NAGNAG acceptors are overrepresented in genes coding for proteins with RNA-recognition motifs (RRMs). As SR proteins, a family of RRM-containing important splicing factors, are known to be extensively alternatively spliced in Arabidopsis, we analyzed alternative splicing at NAGNAG acceptors in SR and SR-related genes.ResultsIn a comprehensive analysis of the Arabidopsis thaliana genome, we identified 6,772 introns that exhibit a NAGNAG acceptor motif. Alternative splicing at these acceptors was assessed using available EST data, complemented by a sequence-based prediction method. Of the 36 identified introns within 30 SR and SR-related protein-coding genes that have a NAGNAG acceptor, we selected 15 candidates for an experimental analysis of alternative splicing under several conditions. We provide experimental evidence for 8 of these candidates being alternatively spliced. Quantifying the ratio of NAGNAG-derived splice variants under several conditions, we found organ-specific splicing ratios in adult plants and changes in seedlings of different ages. Splicing ratio changes were observed in response to heat shock and most strikingly, cold shock. Interestingly, the patterns of differential splicing ratios are similar for all analyzed genes.ConclusionNAGNAG acceptors frequently occur in the Arabidopsis genome and are particularly prevalent in SR and SR-related protein-coding genes. A lack of extensive EST coverage can be compensated by using the proposed sequence-based method to predict alternative splicing at these acceptors. Our findings indicate that the differential effects on NAGNAG alternative splicing in SR and SR-related genes are organ- and condition-specific rather than gene-specific.

The 3′ Untranslated Region of the Rabies Virus Glycoprotein mRNA Specifically Interacts with Cellular PCBP2 Protein and Promotes Transcript Stability

Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3′ UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3′ UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

Differential Recruitment of Splice Variants from SR Pre-mRNAs to Polysomes During Development and in Response to Stresses

We have previously shown that precursor mRNAs (pre-mRNAs) of serine/arginine-rich (SR) proteins are extensively alternatively spliced to generate approximately 100 distinct splice variants from 14 SR genes and that the splicing pattern of SR pre-mRNAs changes in different organs and in response to abiotic stresses. About half of the splice variants are potential targets of nonsense-mediated decay (NMD) and 25 splice forms were confirmed to be real NMD targets. However, it is not known whether (i) all splice variants are recruited to polysomes for translation; (ii) there is a preferential recruitment of specific splice isoforms to polysomes; and (iii) there is a differential recruitment of splice variants during development and in response to stresses. To address these questions, we analyzed the association of SR splice variants with polysomes from seedlings, different organs and seedlings exposed to heat and cold stress. In seedlings, about one-third of the splice variants (22 out of 72) are not recruited to polysomes. Among those associated with polysomes, the functional isoforms that code for full-length proteins and some candidate putative and confirmed NMD targets were identified. There was preferential recruitment of some splice forms over others. Predominant recruitment of functional isoforms along with a few NMD candidates was found in different organs. Furthermore, we observed differential recruitment of isoforms in different organs. Heat and cold stress enhanced or reduced recruitment of specific splice variants. Our studies reveal differential recruitment of SR splice variants to polysomes under normal conditions, during development and in response to stresses.

Approaches for the Identification and Characterization of RNA-Protein Interactions

Based on analyses of conventional RNA binding motifs, there are well over 550 RNA binding proteins produced in living cells. Thus a detailed understanding of the mechanisms of post-transcriptional regulation requires an understanding of how these trans-acting factors act in both local and combinatorial fashions. The goal of this chapter is to provide a roadmap for approaching the identification of RNA–protein interactions and how to decipher fundamental aspects of their roles in the regulation of gene expression. Many of the techniques outlined below are applicable to the study of RNA–RNA interactions as well. Collectively they should provide a rational approach to gain mechanistic insights into a variety of post-transcriptional processes.