Son maintains accurate splicing for a subset of human pre-mRNAs

Abstract

Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

Btf and TRAP150 have distinct roles in regulating subcellular mRNA distribution

“…In contrast to what was previously observed following depletion of other splicing factors, 16 we did not observe altered splicing of BTM transcripts after depletion of Btf or TRAP150 (data not shown). Since Btf depletion caused a dramatic difference in cytoplasmic levels of BTM transcripts, we looked for changes in recruitment of other mRNA processing factors to the BTM locus following Btf depletion.…”

“…2a) that is constitutively active and can be visualized by RNA fluorescence in situ hybridization (FISH) as a single transcription locus in each cell nucleus. 15,16 BTM HeLa cells were processed for localization of BTM transcripts by RNA-FISH, followed by immunolabeling of splicing factor SRSF1 and either Btf or TRAP150. In contrast to the limited colocalization observed between Btf or TRAP150 with SRSF1 at nuclear speckles, both Btf and TRAP150 colocalized with SRSF1 at the BTM transcription site ( fig.…”

“…We conclude that while these two proteins have several features in common, their functions are not entirely panels f, p and z). Seventy-two hours after knockdown, the cells were processed for RNA-FISH localization of reporter RNA using oligos against exon 5 (panels b, g, l, q, v and aa) as described 16 and immunofluorescence localization of Btf, splicing factor SF2/ ASF or GFP. Arrows indicate the BTM transcription site.…”

“…RNA-FISH was performed as described earlier. 13,15,16,32 Cells were fixed with 2% formaldehyde for 15 min at room temperature followed by three washes with PBS, 5 min each. Extraction was performed in cytoskeleton (CSK) extraction buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 10 mM PIPES pH 6.8, 625 μl of 0.5% Triton-X-100, 2 mM VRC).…”

Imaging Cellular Metabolism

“…A well-characterized beta-tropomyosin (BTM) minigene (Helfman et al, 1988;Huang & Spector, 1996) was stably integrated into HeLa cells (SaccoBubulya and Spector, 2002;Sharma et al, 2011). The BTM minigene is useful for monitoring constitutive as well as alternative splicing of minigene transcripts.…”