Abstract
Nuclear speckles are subnuclear storage sites containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as serine/arginine-rich splicing factor 1 (SRSF1 or ASF/SF2) and SRSF2 (SC35). NSrp70 mediates alternative splice site selection, targeting several pre-mRNAs, including CD44 exon v5. Here we demonstrated that NSrp70 interacts physically with two SR proteins, SRSF1 and SRSF2, and reverses their splicing activity in terms of CD44 exon v5 as exon exclusion. The NSrp70 RS-like region was subdivided into three areas. Deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins and to target mRNA and also failed to induce splicing of CD44 exon v5, suggesting that RS1 is critical for NSrp70 functioning. Interestingly, RS1 deletion also resulted in the loss of NSrp70 and SR protein speckle positioning, implying a potential scaffolding role for NSrp70 in nuclear speckles. NSrp70 contains an N-terminal coiled-coil domain that is critical not only for self-oligomerization but also for splicing activity. Consistently, deletion of the coiled-coil domain resulted in indefinite formation of nuclear speckles. Collectively, these results demonstrate that NSrp70 acts as a new molecular counterpart for alternative splicing of target RNA, counteracting SRSF1 and SRSF2 splicing activity.
Highlights
The mammalian cell nucleus contains non-membranous subnuclear bodies such as the nucleolus, Cajal bodies, promyelocytic leukemia bodies, and nuclear speckles
We have previously identified nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as serine/arginine-rich splicing factor 1 (SRSF1 or ASF/SF2) and SRSF2 (SC35)
NSrp70 Counteracts SRSF1- and SRSF2-induced CD44 Exon v5 Exclusion—We have reported previously that NSrp70 modulates alternative splice site selection of some minigenes, including Tra21, Fas, and CD44
Summary
Reagents and Antibodies—Anti-human NSrp (CCDC55) antibody, glutaraldehyde solution, and His-selected nickel affinity gel were purchased from Sigma-Aldrich 48 h after transfection, cells were collected for RNA and protein purification or image analysis. RNA Immunoprecipitation—RNA immunoprecipitation (RIP) experiments were performed using a Magna RIP RNAbinding protein immunoprecipitation kit according to the instructions of the manufacturer (Millipore) using a modified version of a method described previously [18]. HEK293T cells (1 ϫ 107) transfected with the indicated constructs were lysed on ice in complete RIP lysis buffer (Millipore). Protein lysates were incubated with rabbit anti-NSrp70- or rabbit control IgG-coated beads overnight at 4 °C. The RNA-protein complexes were dissociated from the beads by incubating in proteinase K buffer (Millipore) for 30 min at 55 °C. Protein Cross-linking Assay—For cross-linking of cell lysates, cells were transfected with the indicated plasmids and lysed with lysis buffer as described above. Differences between groups were considered significant at p Ͻ 0.05
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