Smu1 and RED are required for activation of spliceosomal B complexes assembled on short introns

Panagiotis Papasaikas,Cindy L Will,Juan Valcárcel,Sandra Keiper,Reinhard Lührmann,Cyrille Girard

doi:10.1038/s41467-019-11293-8

Abstract

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.

Highlights

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes
To elucidate the role of Smu[1] and RED, we investigated global effects on pre-mRNA splicing after their knockdown in HeLa cells using RNAi followed by RNA-Seq analysis
Consistent with previous results obtained using multiplexed reverse transcription polymerase chain reaction (RT-PCR) analysed by capillary electrophoresis (LabChip)[15], Smu[1] or RED depletion led to changes in the alternative splicing (AS) of cassette exons (Cex) (Fig. 1a), the majority of which were skipped (Supplementary Fig. 1c) or in the selection of alternative 5′ and 3′ splice sites (Fig. 1a)

Summary

Introduction

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. In vitro splicing assays reveal that Smu[1] and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. The recent cryogenic electron microscopy (cryo-EM) structure of the human B complex shed light on possible roles for Smu[1] and RED, indicating that they play an important structural role in bridging Brr[2] with the U2 protein SF3B329 This observation challenges the notion that RED and Smu[1] solely play regulatory roles, suggesting instead a core structural function during spliceosome assembly

Methods

Results

Conclusion