Identification of gene expression and splicing changes upon eye-specific downregulation of tri-snRNP components in Drosophila

The spliceosome is a dynamic RNA-protein complex that executes pre-mRNA splicing and is composed of five core small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and >150 additional proteins specific for each snRNP. We report a circadian role for Pre-mRNA Processing factor 4 (PRP4), a conserved component of the spliceosomal U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. We broadly hypothesized that downregulation of prp4 led to the aberrant splicing of one or many of the core clock transcripts. To identify these splicing events in an unbiased way, we performed RNA-Sequencing (RNA-Seq) analysis. We reasoned that we could have a more targeted approach if we could zoom in on the overlapping splicing changes that would be driven by the knockdown of at least two different tri-snRNP components. Because the pan-neuronal knockdown of all tri-snRNP components tested in our study led to lethality, we decided to utilize an alternative broad driver. For that purpose, we selected a strong eye-specific Glass Multiple Promoter driver (GMR-Gal4). Because most of the signal from head lysates comes directly from the eye tissue and because the core splicing factors are ubiquitously expressed, GMR-specific downregulation of prp4 and prp8 promised to be a viable alternative to the pan-neuronal knockdown. We examined changes in both the total transcript levels and splicing events upon prp4 knockdown in the eye. The overall gene expression seemed to be dramatically influenced by prp4 downregulation (433 DOWN, 310 UP at FDR < 0.05). Despite the fact that PRP4 is a component of the core spliceosome that is required for constitutive exon splicing, we did not detect dramatic effects on global splicing. Only 45 genes exhibited differential alternate splicing upon prp4 downregulation at FDR < 0.05). Overall design: 3 samples with 5 replicates each were analyzed using Illumina Next-Generation Sequencing (NextSeq 500).

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Shakhmantsir I, Nayak S, Grant GR, Sehgal A