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.

Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis.

Pectate lyases catalyse the eliminative cleavage of de-esterified homogalacturonan in pectin, a major component of the primary cell walls in higher plants. In the completed genome of Arabidopsis, there are 26 genes (AtPLLs) that encode pectate lyase-like proteins. Here, we analysed the expression pattern of all AtPLLs in different organs, at different stages of seedling development and in response to various hormones and stresses. The expression of PLLs varied considerably in different organs, with no expression of some PLLs in vegetative organs. Interestingly, all PLL genes are expressed in flowers. Several PLLs are expressed highly in pollen, suggesting a role for these in pollen development and/or function. Analysis of expression of all PLL genes in seedlings treated with hormones, abiotic stresses and elicitors of defense responses revealed significant changes in the expression of some PLLs without affecting the other PLLs. The stability of transcripts of PLLs varied considerably among different genes. Our results indicate a complex regulation of expression of PLLs and involvement of PLLs in some of the hormonal and stress responses.

Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses.

Precursor mRNAs with introns can undergo alternative splicing (AS) to produce structurally and functionally different proteins from the same gene. Here, we show that the pre-mRNAs of Arabidopsis genes that encode serine/arginine-rich (SR) proteins, a conserved family of splicing regulators in eukaryotes, are extensively alternatively spliced. Remarkably about 95 transcripts are produced from only 15 genes, thereby increasing the complexity of the SR gene family transcriptome by six-fold. The AS of some SR genes is controlled in a developmental and tissue-specific manner. Interestingly, among the various hormones and abiotic stresses tested, temperature stress (cold and heat) dramatically altered the AS of pre-mRNAs of several SR genes, whereas hormones altered the splicing of only three SR genes. These results indicate that abiotic stresses regulate the AS of the pre-mRNAs of SR genes to produce different isoforms of SR proteins that are likely to have altered function(s) in pre-mRNA splicing. Sequence analysis of splice variants revealed that predicted proteins from a majority of these variants either lack one or more modular domains or contain truncated domains. Because of the modular nature of the various domains in SR proteins, the proteins produced from splice variants are likely to have distinct functions. Together our results indicate that Arabidopsis SR genes generate surprisingly large transcriptome complexity, which is altered by stresses and hormones.