Crystal structure of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain

Abstract

Spliceosomal proteins Hsh49p and Cus1p are components of SF3b, which together with SF3a, Msl1p/Lea1p, Sm proteins, and U2 snRNA, form U2 snRNP, which plays a crucial role in pre-mRNA splicing. Hsh49p, comprising two RRMs, forms a heterodimer with Cus1p. We determined the crystal structures of Saccharomyces cerevisiae full-length Hsh49p as well as its RRM1 in complex with a minimal binding region of Cus1p (residues 290-368). The structures show that the Cus1 fragment binds to the α-helical surface of Hsh49p RRM1, opposite the four-stranded β-sheet, leaving the canonical RNA-binding surface available to bind RNA. Hsh49p binds the 5 ′ end region of U2 snRNA via RRM1. Its affinity is increased in complex with Cus1(290-368)p, partly because an extended RNA-binding surface forms across the protein-protein interface. The Hsh49p RRM1-Cus1(290-368)p structure fits well into cryo-EM density of the B act spliceosome, corroborating the biological relevance of our crystal structure.

Arabidopsis JANUS Regulates Embryonic Pattern Formation through Pol II-Mediated Transcription of WOX2 and PIN7

“…First, JANUS is critical for the transcription but not RNA splicing of WOX2 and PIN7 , two genes critical for early embryonic pattern formation. Second, the RRM1 domain of yeast SAP49 or metazoan Hsh49p, homolog of Arabidopsis JANUS, is responsible for RNA binding and spliceosome association and is essential for spliceosome-mediated RNA splicing (Igel et al., 1998, Kuwasako et al., 2017, Pauling et al., 2000, van Roon et al., 2017). However, we found that JANUS directly interacts with Pol II through its RRM2 but not RRM1 domain, suggesting that its interaction with Pol II does not require its association with the spliceosome.…”

“…However, we found that JANUS directly interacts with Pol II through its RRM2 but not RRM1 domain, suggesting that its interaction with Pol II does not require its association with the spliceosome. Third, yeast and metazoan homologs of JANUS are part of an SF3b complex within the spliceosome (Kuwasako et al., 2017, van Roon et al., 2017). However, although other components of the SF3b complex in Arabidopsis have been functionally characterized (Aki et al., 2011, Wang and Brendel, 2006), none of the related mutants showed embryo lethality.…”

Structure of the human activated spliceosome in three conformational states

“…SF3b145 was generated from Cus1 of the yeast B act complex. The N-terminal domain of SF3b145 and the RRM domain of SF3b49 was generated from the crystal structure of Hsh49p in complex with Cus1p (PDB code: 5LSB 64 ). Crystal structure of SF3b14a/p14 in complex with SF3b155 N-terminal fragments (PDB code: 2F9J 65 ) was docked into the SF3b region of the cryo-EM maps.…”

A Challenging Pie to Splice: Drugging the Spliceosome

“…One of the most impressive recent advances has been the development of detailed structures of specific spliceosome complexes using a combination of cryo-EM[46] and X-ray crystallography. [47] Cryo-EM has proven particularly useful to tease out the organization of the snRNPs within each complex and illustrate the structural rearrangements within each snRNP. While outside the scope of this review, structures of spliceosome complexes that include the U1 snRNP, [48] complex A, [49] the U4/U5/U6 complex, [50] complex C,[51] and the post-spliceosome complex[52] are beginning to reveal the mechanistic motions within this machine.…”