A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence

Abstract

The DEAD-box RNA helicase Prp5 is required for the formation of the prespliceosome through an ATP-dependent function to remodel U2 small nuclear ribonucleoprotein particles (snRNPs) and an ATP-independent function of unknown mechanism. Prp5 has also been implicated in proofreading the branch site sequence, but the molecular mechanism has not been well characterized. Using actin precursor mRNA (pre-mRNA) carrying branch site mutations, we identified a Prp5-containing prespliceosome with Prp5 directly bound to U2 small nuclear RNA (snRNA). Prp5 is in contact with U2 in regions on and near the branchpoint-interacting stem-loop (BSL), suggesting that Prp5 may function in stabilizing the BSL. Regardless of its ATPase activity, Prp5 mutants that suppress branch site mutations associate with the spliceosome less tightly and allow more tri-snRNP binding for the reaction to proceed. Our results suggest a novel mechanism for how Prp5 functions in prespliceosome formation and proofreading of the branch site sequence. Prp5 binds to the spliceosome in association with U2 by interacting with the BSL and is released upon the base-pairing of U2 with the branch site to allow the recruitment of the tri-snRNP. Mutations impairing U2-branch site base-pairing retard Prp5 release and impede tri-snRNP association. Prp5 mutations that destabilize the Prp5-U2 interaction suppress branch site mutations by allowing progression of the pathway.[Keywords: Prp5; splicing; branch site; U2; proofreading] Supplemental material is available for this article.Received October 4, 2014; revised version accepted November 10, 2014.Introns of precursor mRNAs (pre-mRNAs) are removed via two steps of transesterification reaction, catalyzed by the spliceosome, which consists of five small nuclear RNAs (snRNAs; U1, U2, U4/U6, U5) and many protein factors. The snRNAs, each complexed with a number of proteins to form a small nuclear ribonucleoprotein particle (snRNP), bind to the pre-mRNA sequentially in an order of U1 and U2 and then U4/U6.U5 as a preformed trisnRNP (Brow 2002;Wahl et al. 2009;Will and L€ uhrmann 2011). Spliceosome assembly initiates with the binding of U1 to the 59 splice, forming the commitment complex (CC), followed by binding of U2 to the branch site to form the prespliceosome. Respective recognition of the 59 splice site and the branch site by U1 and U2 is mediated through RNA base-pairing interactions between the snRNAs and the intronic sequence. Prior to U2 addition, a Msl5-Mud2 heterodimer binds to the branch site and interacts with U1 snRNP, bridging the interaction between the 59 splice site and the branch site (Berglund et al. 1997(Berglund et al. , 1998Wang et al. 2008). Msl5-Mud2 is displaced from the branch site before U2 snRNA can base-pair with the branch site sequence in the formation of the prespliceosome. Following binding of the tri-snRNP, the spliceosome undergoes a major structural rearrangement to release U1 and U4 and forms new base pairs between U6 and the 59 splice site and between U2 and U6. A protein complex assoc...

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

“…As expected, in the presence of wild-type Hsh155p, wild-type Flag-Prp5p efficiently coimmunoprecipitated pre-mRNA, U1 and U2 snRNAs, and much less U4, U5, or U6 snRNA (Fig. 6C), consistent with the previous report that Prp5p is released before the U4/U6.U5 tri-snRNP recruitment (Liang and Cheng 2015). However, wild-type Flag-Prp5p coimmunoprecipitated much less U1 and U2 snRNAs in the presence of hsh155 mutant allele hsh155-L313S or hsh155-H331R, but similar levels of U1 and U2 snRNAs in the presence of the hsh155-H331D or hsh155-K335N allele (Fig.…”

“…Increased Prp5p-Hsh155p interaction leads to an increased release of Prp5p from the prespliceosome Prp5p was demonstrated to be essential for prespliceosome assembly (Xu et al 2004) and could be released immediately after this assembly to allow recruitment of U4/ 5/6 tri-snRNP (Liang and Cheng 2015). To investigate the functional contribution of Prp5p-Hsh155p interaction in prespliceosome assembly, we tested in vivo interactions between Hsh155p and Prp5p proteins in budding yeast lysates by coimmunoprecipitation using HA-tagged hsh155 or Flag-tagged prp5 allele strains.…”

“…Prp5 interacts with U1 snRNP through its N-terminal SR-like domain and with the U2 snRNP SF3B complex through its DPLD motif, providing a U1:Prp5:U2 platform for prespliceosome formation (Xu et al 2004;Shao et al 2012). Mutations in both the ATPase domain and the DPLD motif of Prp5 modulate splicing fidelity, specifically favoring the recognition and use of suboptimal substrates at the BS region (Xu and Query 2007;Liang and Cheng 2015). SF3B1 mutations affect alternative splicing by promoting the use of alternate branch points (Furney Figure 1.…”

Conformational dynamics of stem II of the U2 snRNA

“…How stem IIa and IIc interconvert during the catalytic stages of splicing is also unclear. It has been speculated that Prp5p could be involved in this transition (Hilliker et al 2007;Perriman and Ares 2007;Kosowski et al 2009); however, recent work suggests that there is no obligate role for Prp5p after prespliceosome formation (Liang and Cheng 2015).…”

“…This was based on coimmunoprecipitation (co-IP) assays in which Cus2p coimmunoprecipitated U2 snRNAs with destabilized stem IIa more efficiently than those in which stem IIc formation was prevented (Perriman and Ares 2007). In both models, Prp5p could then displace Cus2p and stabilize the stem IIa conformation prior to branchsite duplex formation (Liang and Cheng 2015). It is not clear if Prp5p may also actively destabilize stem IIc, facilitate annealing of stem IIa, or both.…”