SummaryThere are ∼650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease.

SummaryAlternative inclusion of exons increases the functional diversity of proteins. Among alternatively spliced exons, tissue-specific exons play a critical role in maintaining tissue identity. This raises the question of how tissue-specific protein-coding exons influence protein function. Here we investigate the structural, functional, interaction, and evolutionary properties of constitutive, tissue-specific, and other alternative exons in human. We find that tissue-specific protein segments often contain disordered regions, are enriched in posttranslational modification sites, and frequently embed conserved binding motifs. Furthermore, genes containing tissue-specific exons tend to occupy central positions in interaction networks and display distinct interaction partners in the respective tissues, and are enriched in signaling, development, and disease genes. Based on these findings, we propose that tissue-specific inclusion of disordered segments that contain binding motifs rewires interaction networks and signaling pathways. In this way, tissue-specific splicing may contribute to functional versatility of proteins and increases the diversity of interaction networks across tissues.

SummaryPoly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.

DNA in the eukaryotic nucleus is packaged in the form of nucleosomes, ~147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP dependent chromatin remodelers1–3 such as the 15 subunit INO80 complex4. INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, exchanging histone H2A.Z with H2A, and positioning +1 and -1 nucleosomes at promoter DNA5–8. A structure and mechanism for these remodeling reactions is lacking. Here we report the cryo-electron microscopy structure at 4.3Å resolution, with parts at 3.7Å, of an evolutionary conserved core INO80 complex from Chaetomium thermophilum bound to a nucleosome. INO80core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. A Rvb1/2 AAA+ ATPase hetero-hexamer is an assembly scaffold for the complex and acts as stator for the motor and nucleosome gripping subunits. The Swi2/Snf2 ATPase motor binds to SHL-6, unwraps ~15 base pairs, disrupts the H2A:DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5-Ies6 grip SHL-2/-3 acting as counter grip for the motor on the other side of the H2A/H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 core and entry DNA over a distance of ~90Å and packs against histone H2A/H2B near the acidic patch. Our structure together with biochemical data8 suggest a unified mechanism for nucleosome sliding and histone editing by INO80. The motor pumps entry DNA across H2A/H2B against Arp5 and the grappler, sliding nucleosomes as a ratchet. Transient exposure of H2A/H2B by the motor and differential recognition of H2A.Z and H2A may regulate histone exchange during translocation.