Berthold Kastner

Formation of the activated human spliceosome (B ^act ) involves major structural rearrangements, leading to the catalytically active U2/U6 RNA core. This process involves at least two intermediates, pre-B ^act-1 and pre-B ^act-2 , and is regulated by CDK11-mediated phosphorylation of the U2 snRNP protein SF3B1. However, the mechanisms of this essential step are poorly understood. Here we present the cryo-EM structure of a spliceosome stalled – by the CDK11 inhibitor OTS964 – in a previously undescribed early-activated state, termed pre-B ^act-OTS , shortly after dissociation of U4 snRNP. In pre-B ^act-OTS , the U2-SF3B6 protein is retained in a C-terminal region of the super-helical U2-SF3B1 HEAT domain (SF3B1 ^HEAT ) that clamps the U2/branch-site helix. In contrast, in pre-B ^act-1 , SF3B6 is repositioned to SF3B1’s N-terminal HEAT repeats, thereby preventing a steric clash of SF3B6 with PRP8 during the pre-B ^act-OTS -to-pre-B ^act-1 transition. We infer that the CDK11-mediated phosphorylation of SF3B1 drives the relocation of SF3B6, gating progression to B ^act formation. In pre-B ^act-OTS , we also located the RNA helicase DHX15 at the N-terminal region of SF3B1 ^HEAT , assisted by the SR140/SPF45/CHERP/SUGP1 protein complex. These results suggest the involvement of DHX15 in kinase-mediated proofreading of the early-activated spliceosome, by competing with CDK11’s phosphorylation of SF3B1, and thus with relocation of SF3B6 at SF3B1 ^HEAT .

Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3′ splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA–mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3′ss. Cryo–electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3′ region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3′ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3′ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3′ss choices.

Splicing machine shifts into gear: Spliceosome activation involves extensive protein exchanges and RNA rearrangements that lead to the formation of a catalytically active U2/U6 RNA structure called B ^act . Previously, little was known about the pathway leading to the U2/U6 active site and how proteins aid in folding the U2/U6 RNA. Using cryo–electron microscopy to determine structures of two human pre-B ^act complexes, Townsend et al. uncovered an intricate cascade of coordinated structural changes involving mutually exclusive interactions that facilitate the directionality of the activation process. These structures reveal the assembly pathway of the U2/U6 catalytic RNA and the mechanism whereby proteins facilitate its folding. Science , this issue p. eabc3753

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition intransof an excess of 5′ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5′ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5′ss sequence, most importantly between Prp8 and nucleotides at the exon–intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.

Previous in vitro studies have demonstrated that enzyme proteins liberated from isolated zymogen granules of the rat pancreas aggregate already at neutral or slightly basic pH and form small particles which in the acidic pH range progressively condense into dense cores of about the size of zymogen granules. To characterize the protein composition of the original particles in more detail non-denaturing agarose gel electrophoresis was employed. Five major protein complexes were identified which upon separation of individual complexes in 1-D or 2-D gel electrophoresis were shown to be composed of a distinct set of known enzymes and several unknown proteins. Complexes 1–4 quickly dissociated when enzyme activation was induced by enterokinase, but complex 5 was resistent even to this treatment. All 5 complexes revealed a distinct fine structure when eluted from the gels and studied in negative staining electron microscopy. These findings suggest that pancreatic zymogens form complexes already in the lumen of the rough endoplasmic reticulum and are transported as such to the Golgi complex where they aggregate into granule cores due to the internal acidic pH. Complex formation may thus facilitate zymogen sorting within the rough endoplasmic reticulum and may prevent premature enzyme activation within cellular compartments.