Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences

Keiko Horiuchi,Takeshi Kawamura,Takao Hamakubo

doi:10.1016/j.jbc.2021.101248

Abstract

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

Highlights

Alternative splicing (AS) is the process by which a single gene produces multiple mRNAs, thereby substantially increasing both the protein diversity and complexity
Gene ontology (GO) analysis of the differentially expressed genes (DEGs) showed enrichment of cellular processes linked to cell cycle, immune response, morphogenesis, and cell migration by Wilms’ tumor 1-associating protein (WTAP), cell cycle and chemokine secretion by VIRMA, cell migration and the metabolic process by ZC3H13, and cell cycle, cell adhesion and cell migration by CBLL1 (Supplementary Figure S2)
Heatmap of the WTAP-regulated genes showed that majority of genes are regulated by KD of VIRMA (74.7%), ZC3H13 (86.2%) and CBLL1 (73.0%) as well as KD of other WTAP-interacting proteins BCLAF1/THRAP3 (70.8%), RBM15/RBM15B (54.9%), METTL3 (57.4%), and METTL14 (59.0%) (Figure 1D and Supplementary Figure S3A). (Note, a similar trend was inferred when the change in gene expression was in the same direction as that of WTAP-KD and |Log2FC| >= 0.1)

Summary

INTRODUCTION

Alternative splicing (AS) is the process by which a single gene produces multiple mRNAs, thereby substantially increasing both the protein diversity and complexity. Recent studies have shown that m6A modification in mRNAs or non-coding RNAs influences multiple cellular processes in various organisms, including sex determination and dosage compensation in Drosophila and X chromosome inactivation in mammals, as well as viability, meiosis, tissue development, maternal-to-zygotic transition, stem cell self-renewal and differentiation, HIV replication, circadian. Our previous proteomic study using HUVEC whole cell lysates showed that WTAP forms a protein complex comprising Virilizer homolog (VIRMA), CBLL1 (Hakai), ZC3H13(KIAA0853), which were identified as the major components isolated from all of the three different anti-WTAP antibodies with high total spectrum count in LC/MS/MS [15], and RBM15, as well as METTL3 and METTL14. Since our previous proteomic data revealed an evolutionarily conserved formation of the WTAP complex and its interaction with general splicing factors [15], here, we investigate whether the WTAP complex regulates AS in mammalian cells, independent or dependent on the m6A modifications. Further analysis of the sequences in the regulated introns/exons, as well as minigene analysis, revealed the involvement of G-quadruplex formation near 5` and 3` splice sites in AS regulation by WTAP, suggesting possible participation of the G-quadruplex-interacting/WTAP-interacting proteins, such as FMR1, FXR1, and FXR2

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES