Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex

Abstract

SF3b1 is an essential component of the U2 snRNP implicated in branch site (BS) recognition and found to be frequently mutated in several human cancers. While recent structures of yeast and human SF3b1 have revealed its molecular architecture, the importance of specific RNA:protein contacts and conformational changes remains largely uncharacterized. Here, we performed mutational analysis of yeast SF3b1, guided by recent structures of the spliceosome. We find that conserved amino acids contacting the U2 snRNA backbone of the U2/BS duplex are nonessential, and that yeast can tolerate truncation of the HEAT repeats containing these amino acids. The pocket housing the branchpoint adenosine (BP-A) is also amenable to mutation despite strong conservation. However, mutations that support viability can still lead to defects in splicing pre-mRNAs with nonconsensus BS substitutions found at -3, -2, -1, and +1 positions relative to the BP-A or at the branchpoint position. Through the generation of yeast and human chimeric proteins, we further defined the functionally conserved regions of Hsh155 as well as identify changes in BS usage resulting from inclusion of human SF3b1 HEAT repeats. Moreover, these chimeric proteins confer a sensitivity to small molecule inhibition by pladienolide B to yeast splicing. Together, these data reveal the importance of individual contacts of Hsh155/SF3b1 to the U2/BS duplex and define their contribution to BS usage by the spliceosome.

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

“…1D) are altered to block Cus2 binding. Since deletion of CUS2 is not lethal (Yan et al 1998), we anticipated that the loss of its binding to Hsh155 would also not be lethal, and a recent analysis of Hsh155 deletions in this region is consistent with this (Carrocci et al 2018).…”

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM–ULM interaction

“…1D) are altered to block Cus2 binding. Since deletion of CUS2 is not lethal (Yan et al 1998), we anticipated that the loss of its binding to Hsh155 would also not be lethal, and a recent analysis of Hsh155 deletions in this region is consistent with this (Carrocci et al 2018). Successful CRISPR/Cas9 editing of the RW-DA mutation into the chromosomal copy of HSH155 creates a viable strain with no obvious growth defect at a variety of different temperatures (Supplemental Fig.…”

Silencing Core Spliceosome Sm Gene Expression Induces a Cytotoxic Splicing Switch in the Proteasome Subunit Beta 3 mRNA in Non-Small Cell Lung Cancer Cells

“…In contrast, SF3B1 is part of only the U2 snRNP subunit that participates in every part of the splicing reaction, except the initial step. SF3B1 accommodates the adenosine of the branchpoint sequence in the intronic sequence to support duplex formation with the U2 snRNA [ 20 ]. It is unclear whether this difference in the structural versus dynamic functions of SmD3 and SF3B1 can account for the observed difference in the number of DE and AS events upon their silencing.…”