The intronic branch point sequence is under strong evolutionary constraint in the bovine and human genome

The branch point sequence is a cis-acting intronic motif required for mRNA splicing. Despite their functional importance, branch point sequences are not routinely annotated. Here we predict branch point sequences in 179,476 bovine introns and investigate their variability using a catalogue of 29.4 million variants detected in 266 cattle genomes. We localize the bovine branch point within a degenerate heptamer “nnyTrAy”. An adenine residue at position 6, that acts as branch point, and a thymine residue at position 4 of the heptamer are more strongly depleted for mutations than coding sequences suggesting extreme purifying selection. We provide evidence that mutations affecting these evolutionarily constrained residues lead to alternative splicing. We confirm evolutionary constraints on branch point sequences using a catalogue of 115 million SNPs established from 3,942 human genomes of the gnomAD database.

Investigating the Molecular Mechanism of H3B-8800: A Splicing Modulator Inducing Preferential Lethality in Spliceosome-Mutant Cancers

The SF3B1 protein, part of the SF3b complex, recognizes the intron branch point sequence of precursor messenger RNA (pre-mRNA), thus contributing to splicing fidelity. SF3B1 is frequently mutated in cancer and is the target of distinct families of splicing modulators (SMs). Among these, H3B-8800 is of particular interest, as it induces preferential lethality in cancer cells bearing the frequent and highly pathogenic K700E SF3B1 mutation. Despite the potential of H3B-8800 to treat myeloid leukemia and other cancer types hallmarked by SF3B1 mutations, the molecular mechanism underlying its preferential lethality towards spliceosome-mutant cancer cells remains elusive. Here, microsecond-long all-atom simulations addressed the binding/dissociation mechanism of H3B-8800 to wild type and K700E SF3B1-containing SF3b (K700ESB3b) complexes at the atomic level, unlocking that the K700E mutation little affects the thermodynamics and kinetic traits of H3B-8800 binding. This supports the hypothesis that the selectivity of H3B-8800 towards mutant cancer cells is unrelated to its preferential targeting of K700ESB3b. Nevertheless, this set of simulations discloses that the K700E mutation and H3B-8800 binding affect the overall SF3b internal motion, which in turn may influence the way SF3b interacts with other spliceosome components. Finally, we unveil the existence of a putative druggable SF3b pocket in the vicinity of K700E that could be harnessed in future rational drug-discovery efforts to specifically target mutant SF3b.

A Model for DHX15 Mediated Disassembly of A-Complex Spliceosomes

A critical step of pre-mRNA splicing is the recruitment of U2 snRNP to the branch point sequence of an intron. U2 snRNP conformation changes extensively during branch helix formation and several RNA-dependent-ATPases are implicated in the process. However, the molecular mechanisms involved remain to be fully dissected. We took advantage of the differential nucleotide triphosphates requirements for DExD/H-box enzymes to probe their contributions to in vitro spliceosome assembly. Both ATP and GTP hydrolysis support the formation of A-complex, indicating the activity of a DEAH-enzyme because DEAD-enzymes are selective for ATP. We immunodepleted DHX15 to assess its involvement and although splicing efficiency decreases with reduced DHX15, A-complex accumulation incongruently increases. DHX15 depletion also results in the persistence of the atypical ATP-independent interaction between U2 snRNP and a minimal substrate that is otherwise destabilized in the presence of either ATP or GTP. These results lead us to hypothesize that DHX15 plays a quality control function in U2 snRNP's engagement with an intron. In efforts to identify the RNA target of DHX15, we determined that an extended polypyrimidine tract is not necessary for disruption of the atypical interaction between U2 snRNP and the minimal substrate. We also examined U2 snRNA by RNase H digestion and identified nucleotides in the branch binding region that become accessible with both ATP and GTP hydrolysis, again implicating a DEAH-enzyme. Together, our results demonstrate that multiple ATP-dependent rearrangements are likely involved in U2 snRNP addition to the spliceosome and that DHX15 can have an expanded role in splicing.

Quantification of the evolutionary constraint on intronic branch point sequences in the bovine and human genome

We predict the branch point sequence in 180,892 bovine introns and investigate the variability of this cis-acting splicing element at nucleotide resolution. A degenerate heptamer "nnyTrAy" with conserved thymine and adenine residues at positions 4 and 6 constitutes the bovine branch point sequence. This motif is located between 18 and 37 bp upstream of the 3′ splice acceptor site. More than 90% of the introns contain canonical branch point sequence motifs with thymine and adenine residues at positions 4 and 6. Using a catalogue of 29.4 million variants detected in 266 cattle, we show that the conserved thymine and adenine residues are strongly depleted for mutations. These two intronic residues harbor 39% and 41% less variants than coding sequences suggesting extreme purifying selection. The evolutionary constraint is higher at the thymine residue than the branch point adenine itself in heptamers with a canonical branch point sequence motif. We replicate these observations in human branch point sequences with a catalogue of more than 115 million SNPs detected in 3,942 genomes from the gnomAD database. Our study suggests that the evolutionary constraint is stronger on intronic branch point sequences than coding regions in the bovine and human genome.

Structure of the activated human minor spliceosome

The minor spliceosome mediates splicing of the rare but essential U12-type pre-mRNA. Here we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-Å resolution. The 5′-splice site and branch point sequence of the U12-type intron are recognized by U6atac and U12 small nuclear RNA (snRNA), respectively. Five newly identified proteins stabilize the conformation of the catalytic center. The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome. The RBM48/ARMC7 complex binds the γ-monomethyl phosphate cap at the 5′-end of U6atac snRNA. The U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 snRNP. CRIPT stabilizes U12 snRNP. Our study provides a framework for mechanistic understanding of the function of the minor spliceosome.

Identification of herboxidiene features that mediate conformation-dependent SF3B1 interactions to inhibit splicing

ABSTRACTSmall molecules that target the spliceosome SF3B complex are potent inhibitors of cancer cell growth. The compounds affect an early stage of spliceosome assembly when U2 snRNP first engages the branch point sequence of an intron. Recent cryo-EM models of U2 snRNP before and after intron recognition suggest several large-scale rearrangements of RNA and protein interactions involving SF3B. Employing an inactive herboxidiene analog as a competitor with SF3B inhibitors, we present evidence for multiple conformations of SF3B in the U2 snRNP, only some of which are available for productive inhibitor interactions. We propose that both thermodynamics and an ATP-binding event promote the conformation conducive to SF3B inhibitor interactions. However, SF3B inhibitors do not impact an ATP-dependent rearrangement in U2 snRNP that exposes the branch binding sequence for base pairing. We also report extended structure activity relationship analysis of herboxidiene, which identified features of the tetrahydropyran ring that mediate its interactions with SF3B and its ability to interfere with splicing. In combination with structural models of SF3B interactions with inhibitors, our data leads us to extend the model for early spliceosome assembly and inhibitor mechanism. We postulate that interactions between a carboxylic acid substituent of herboxidiene and positively charged SF3B1 sidechains in the inhibitor binding channel are required to maintain inhibitor occupancy and counteract the SF3B transition to a closed state that is promoted by stable U2 snRNA interactions with the intron.

Disclosing the Impact of Carcinogenic SF3b Mutations on Pre-mRNA Recognition Via All-Atom Simulations

The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3’-splice-site (3’SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3’SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing.

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.

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching

SummaryPre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the branching reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 Å away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 Å away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.