RNA Helicase p68 (DDX5) Regulates tau Exon 10 Splicing by Modulating a Stem-Loop Structure at the 5′ Splice Site

Abstract

Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5 splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/ RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5 splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.

The DEAD-box RNA Helicase Dbp2 Connects RNA Quality Control with Repression of Aberrant Transcription

“…In mammals, p68 has been linked to numerous cotranscriptional processing steps and has been suggested to associate with dsRNA both in vitro and in vivo, consistent with the idea that Dbp2 cotranscriptionally modulates RNA structures (34,69,70). Thus, the role of Dbp2 is likely evolutionarily conserved with future studies providing key insights into the biochemical mechanisms in eukaryotic gene regulation.…”

Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing

“…However, in higher eukaryotes, alternative splicing expands the complexity of the proteome in different tissues (56,57), and this is achieved through complex networks of protein-RNA interactions (58 -60). All three ARVCF-interacting proteins were reported to be involved in alternative splicing of specific genes (37,45,59,61). Upon knockdown of ARVCF, alternative splicing of several genes was significantly altered in our study.…”

“…In MYL6, exon 6 is alternatively included (43), and in NDUFV3, exon 3 is changed (44). As an experimental control, we chose a splicing target of p68, Tau, which shows p68-dependent changes of exon 10 inclusion (45). First, we verified whether the mRNAs of the three different genes were detectable in immunoprecipitates using ARVCF-specific antibodies from lysates of untreated HEK cells (Fig.…”

Dual role of the ddx5/ddx17 RNA helicases in the control of the pro-migratory NFAT5 transcription factor

“…It is believed that ddx5 and ddx17 are recruited on target gene promoters by these transcriptional factors and in turn recruit the RNA polymerase II or enzymes with histone acetylase or deacetylase activities (Metivier et al, 2003;Rossow and Janknecht, 2003;Wilson et al, 2004;Janknecht, 2010;Dutertre et al, 2010a;Fuller-Pace and Moore, 2011). In addition, ddx5 and ddx17 copurify with the splicing machinery or spliceosome and can change alternative splicing-site selection in transcripts produced from the H-ras, CD44 and Tau genes Honig et al, 2002;Guil et al, 2003;Camats et al, 2008;Clark et al, 2008;Kar et al, 2011). There are now many reports indicating that these multifunctional proteins have important implications for cancer development, as recently reviewed (Janknecht, 2010;FullerPace and Moore, 2011).…”