Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation

Haroon Butt,Ayman Eid,Magdy M Mahfouz,Sahar Alshareef,Moussa Benhamed,Anireddy S N Reddy,Martin Crespi,Jeremie Bazin

doi:10.1038/s42003-021-02051-y

Abstract

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1GEX1ARESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.

Highlights

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation
We have previously shown that SF3B1 GEX1A RESISTANT 4 (SGR4) is resistant to the GEX1A33
Our analysis indicated that the germination of SGR4 is not affected even at 10 μM GEX1A while

Summary

Introduction

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation This analysis uncovered a set of genes whose intron retention is regulated by both proteins. The SF3B complex is mainly involved in recognition of branch point adenosine (BPA) and promotion of stable interaction of U2 with pre-mRNA13 This complex is a target of natural compounds with antitumor activities, including Herboxidiene (GEX1A)[14], Spliceostatin A (SSA)[15], and Pladienolide B (PB)[16,17]. Structural and biochemical studies have shown that these compounds bind to the SF3B1–PHF5A complex, occupying the pocket of the SF3B complex and blocking SF3B complex binding to the BP18–20 As a result, these splicing modulators perturb BP recognition and selection, preventing the stable formation of the U2 snRNP–BP duplex[18,21]. SF3B1 (SF3b155) is the largest subunit of the SF3B complex and contains an unstructured N-terminal domain (NTD) and C-

Methods

Results

Conclusion