Abstract
RBM10 is an RNA-binding protein that regulates alternative splicing (AS). It localizes to the extra-nucleolar nucleoplasm and S1-1 nuclear bodies (NBs) in the nucleus. We investigated the biological significance of this localization in relation to its molecular function. Our analyses, employing deletion mutants, revealed that RBM10 possesses two S1-1 NB-targeting sequences (NBTSs), one in the KEKE motif region and another in the C2H2 Zn finger (ZnF). These NBTSs act synergistically to localize RBM10 to S1-1 NBs. The C2H2 ZnF not only acts as an NBTS, but is also essential for AS regulation by RBM10. Moreover, RBM10 does not participate in S1-1 NB formation, and without alterations of RBM10 protein levels, its NB-localization changes, increasing as cellular transcriptional activity declines, and vice versa. These results indicate that RBM10 is a transient component of S1-1 NBs and is sequestered in NBs via its NBTSs when cellular transcription decreases. We propose that the C2H2 ZnF exerts its NB-targeting activity when RBM10 is unbound by pre-mRNAs, and that NB-localization of RBM10 is a mechanism to control its AS activity in the nucleus.
Highlights
RBM10 (RNA-binding motif protein 10, previously called S1-1) is an RNA-binding protein [1]
We previously showed that Cy3-labeled ftz model pre-mRNA introduced into HeLa cell nuclei by microinjection is stored in nuclear domains called TIDRs when cells are incubated with actinomycin D (Act D) [20]
NBTS2, which are located in the KEKE motif and the C2 H2 Zn finger (ZnF) region, respectively
Summary
RBM10 (RNA-binding motif protein 10, previously called S1-1) is an RNA-binding protein [1] It regulates alternative splicing (AS) of various pre-mRNAs [2] via exon skipping, as shown for the Drosophila protein Discs large homolog 4 [3], the Notch pathway. RBM10 localizes to nuclear domains called S1-1 NBs and S1-1 granules. S1-1 granules are much smaller and much more abundant, and likely localize to the perichromatin fibrils that comprise transcription and splicing sites [14]. These S1-1 nuclear domains undergo dynamic changes in response to transcriptional states of cells. To better understand RBM10 localization to S1-1 NBs, we identified the S1-1 NBtargeting sequences (NBTSs) in RBM10 that participate in its targeting and localization to S1-1 NBs, and explored the significance of this localization in relation to its AS regulatory function
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