Abstract
TDP-43 regulates cellular levels of Cajal bodies (CBs) that provide platforms for the assembly and RNA modifications of small nuclear ribonucleoproteins (snRNPs) involved in pre-mRNA splicing. Alterations in these snRNPs may be linked to pathogenesis of amyotrophic lateral sclerosis. However, specific roles for TDP-43 in CBs remain unknown. Here, we demonstrate that TDP-43 regulates the CB localization of four UG-rich motif-bearing C/D-box-containing small Cajal body-specific RNAs (C/D scaRNAs; i.e. scaRNA2, 7, 9 and 28) through the direct binding to these scaRNAs. TDP-43 enhances binding of a CB-localizing protein, WD40-repeat protein 79 (WDR79), to a subpopulation of scaRNA2 and scaRNA28; the remaining population of the four C/D scaRNAs was localized to CB-like structures even with WDR79 depletion. Depletion of TDP-43, in contrast, shifted the localization of these C/D scaRNAs, mainly into the nucleolus, as well as destabilizing scaRNA2, and reduced the site-specific 2′-O-methylation of U1 and U2 snRNAs, including at 70A in U1 snRNA and, 19G, 25G, 47U and 61C in U2 snRNA. Collectively, we suggest that TDP-43 and WDR79 have separate roles in determining CB localization of subsets of C/D and H/ACA scaRNAs.
Highlights
The trans-activating response region DNA-binding protein 43 (TARDBP, TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS), and its diseaseassociated mutations cause its accumulation in the cytoplasm of cells in ALS-related lesions in the brain [1,2]
Given that those two scaRNAs have a UG-rich motif, which is a typical TDP-43-binding motif [6], we examined the binding of TDP-43 via this motif by electrophoresis mobility sift assay using (UG)6 oligonucleotide as an RNA antagonist and demonstrated that (UG)6 oligonucleotide interrupted the binding of those scaRNAs to TDP-43 (Figure 1F), suggesting that UGrich motifs in scaRNA2 and scaRNA28 are responsible for TDP-43 binding
As all four of the scaRNAs that have UG-rich motifs belong to the family of C/D scaRNAs (Supplementary Table S4), we suggest that TDP-43 binds to a subset of C/D scaRNAs containing the UG-rich motif
Summary
The trans-activating response region DNA-binding protein 43 (TARDBP, TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS), and its diseaseassociated mutations cause its accumulation in the cytoplasm of cells in ALS-related lesions in the brain [1,2]. TDP-43 is ubiquitously expressed and is shuttled between the nucleus and cytoplasm but resides mostly in the nucleus at steady state [3]. Fibroblast cell lines expressing TDP-43 with an ALScausing mutation originally isolated from ALS patients have about half the number of gems relative to normal cells [19], indicating that TDP-43 is required for maintain-
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