splice-athon

I was looking through pubmed the other day for papers pertaining to transcriptomics, alternatively referred to as RNA-sequencing. This was an assignment for the other course I am taking this semester - Transcriptomics, which is taught by an E. coli-ologist at SRTC. The assignment was straightforward: find a paper in your field pertaining to RNA-seq. I ended up choosing a paper that evaluated the downstream activity of a type II protein arginine methyl transferase (PRMT). These specialized proteins add methyl groups to arginine residues to other proteins, primarily RNA-binding proteins. This particular PRMT had been initially assessed in humans before researchers in my field utilized bioinformatics to find the homologue. The Arabidopsis homologue of PRMT5 was shown to a be a precursor for proper pre-mRNA splicing. The experiments were elegantly designed for the level of complexity that the research possessed, which made this a special read. They mutated AtPRMT5 (inserted ~10kb DNA junk in coding region of gene), extracted total RNA and used this template, along with wild-type (WT), for SOLiD RNA-seq. The results were profound. The transcriptome landscape in the mutant background was quite divergent from that of the WT reads. The scientist looked for mis-spliced transcripts, in other words, transcripts that contained activity through introns. If the mRNA was being properly spliced, there would not be introns present, or there would be no activity through intronic regions. They identified ~14 genes that were mis-spliced. the most important, and phenotypically quantifiable of their findings was the misregulated mRNA splicing of the floral regulator FLK (FLOWERING LOCUS KH DOMAIN) which is a negative regulator of FLC (FLOWERING LOCUS C) which directly down-regulates key floral initiation genes. FLK was shown to be mis-spliced in the prmt5 background, but contained a normal transcript in the WT background. This was confirmed by RNA blot , which showed one band in the WT background, where the mutant background contained splice variants of FLK, consistent with the RNA-seq findings. Next, they examined FLC expression in WT and prmt5 backgrounds. FLC was weakly expressed in the WT background, and strongly expressed in the mutant background. This finding suggests that the mis-splicing of FLK is unable to negatively regulate the expression of FLC, which must be down-regulated to properly transition to the reproductive state. They then examined flowering time of WT and mutant plants. prmt5 plants flowered twice as slow as WT plants due to the misregulation of the floral repressor, FLC. I thought it was both amazing and elegant that such careful experimental design could tie the smallest genetic discrepancy to such a grossly recognizable phenotype. That is all.