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

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

Rapid report Extensive coupling of alternative splicing of pre-mRNAs of serine⁄arginine (SR) genes with nonsense-mediated decay

Summary • 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

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 (premRNAs) 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 fulllength 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

The 3′ UTR of the rabies virus G mRNA represses deadenylation of a reporter RNA in a cell free deadenylation/decay assay.

<p>Panel A: Radiolabeled capped and polyadenylated RNAs containing either reporter only (Gem A60) or the reporter plus the 3′ UTR of the rabies virus P, (P 3′ UTR), M, (M 3′ UTR) or G (G 3′ UTR) mRNAs were incubated with HeLa cytoplasmic extract in a cell-free deadenylation/decay assay. Aliquots were taken at the times indicated and reaction products were analyzed on a 5% polyacrylamide gel containing urea. The position of the polyadenylated (A₆₀) input RNA is indicated in the ‘0’ lane and a deadenylated marker RNA is run in the ‘A₀’ lane. Panel B: Graphical representation of three independent experiments as described in Panel A. The error bars represent standard deviations.</p

The protein responsible for stabilizing reporter RNAs containing the 3′ UTR of the rabies virus G mRNA is a poly(C) binding protein.

<p>Panel A. Radiolabeled, capped and polyadenylated reporter RNA containing the 3′ UTR of the rabies virus G mRNA was incubated in the cell-free RNA deadenylation/decay system using HeLa cytoplasmic extract for 10 minutes in the presence of the indicated amount of poly(C) competitor RNA. Reaction products were analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the gel. The lane marked input indicates the RNA in a reaction that was set up but not incubated. Panel B: Same as panel A. 2.5 µgs of the indicated competitor RNA were added in the lanes marked poly C and poly G. The lane marked ‘0’ indicates a control deadenylation reaction to which no competitor RNA was added. Reactions were incubated for 10 minutes. Panel C: HeLa cytoplasmic extracts were immunodepleted using the indicated antisera. In the left panel, the extent of immunodepletion was examined by western blot using the indicated antisera. GAPDH was used as a normalization control. In the right panel, extracts immunodepleted using either control IgG or PCBP2-specific antibodies were used in our cell-free deadenylation assays with a reporter RNA containing the 3′ UTR of the G mRNA. Samples were taken at the times indicated and analyzed on a 5% polyacrylamide gel containing urea. The positions of the input polyadenylated (A₆₀) and deadenylated product (A₀) are indicated at the left of the RNA gel.</p

The cellular PCBP2 protein selectively interacts with the G mRNA during rabies virus infection.

<p>Rabies virus infected 293T cells were treated with formaldehyde to stabilize RNA-protein complexes and cell lysates were immunoprecipitated using either control IgG or a PCBP2-specific antisera. Co-precipitating rabies viral mRNAs were analyzed by RT-PCR after reversal of the formaldehyde cross links and products run on a 1% agarose gel and visualized with ethidium bromide. The 10% input lane represents total RNA present in the lysate prior to immunoprecipitation. The numbers below the lanes represent quantification and standard deviation from three independent experiments.</p

Electroporated reporter RNAs can be stabilized by a conserved segment of the rabies virus G 3′UTR.

<p>A radiolabeled, capped and polyadenylated reporter RNA derived from pGem-A60 (reporter only), or a variant containing a 72 base segment from the 3′ UTR of the rabies virus G mRNA (reporter+G 3′ UTR), were electroporated into BHK cells. Total RNA was isolated from cells at the times indicated, analyzed on a 5% polyacrylamide gel containing urea, and visualized by phosphorimaging. A graphical representation of the results obtained independent representative experiment is shown. The standard deviation shown in the calculation of half-life differences was determined from 3 independent experiments.</p