Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically-active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here we report the cryo-electron microscopy structures of two human pre-Bact complexes at core resolutions of 3.9-4.2 Å. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually-exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final 3D folding of the U2/U6 catalytic RNA.

Mechanism of 5ʹ splice site transfer for human spliceosome activation

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

Brr2 is a splicing fidelity factor

Many spliceosomal DExD/H box helicases act as fidelity factors during pre-mRNA splicing, promoting on-pathway interactions while simultaneously minimizing errors. Mutations linked to Retinitis Pigmentosa (RP), a form of heritable blindness, map to key domains of spliceosomal helicase Brr2 (SNRNP200 in humans). Previous data show that such mutations negatively impact spliceosome activation, likely due to defects in brr2-RP RNA binding, helicase, and ATPase activities. Furthermore, data from human reporter constructs suggest that brr2-RP might impact 5′ splice site selection. Here we undertake a systematic analysis of brr2-RP effects on splicing fidelity. We show that a subset of brr2-RP mutants exhibit intron retention in vivo. Furthermore, brr2-RP mutants display hyperaccurate and/or error-prone splicing of a variety of splicing reporters. Branch-site fidelity is particularly impacted in this reporter assay. In addition, multiple brr2-RP alleles genetically interact with prp16 alleles known to impact the fidelity of branch site selection. Together these data implicate Brr2 in the fidelity of branch-site selection, and suggest that RP results not just from defects in spliceosome activation, but also from fidelity defects arising throughout the splicing cycle and in splicing fidelity.

Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.