A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.

Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers1-3 such as the 15-subunit INO80 complex 4 . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA5-8. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA+ ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a 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 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data 8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.

Structural mechanism of extranucleosomal DNA readout by the INO80 complex.

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo-electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes.

Genome information processing by the INO80 chromatin remodeler positions nucleosomes.

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin.

Single-molecule nucleosome remodeling by INO80 and effects of histone tails.

Genome maintenance and integrity requires continuous alterations of the compaction state of the chromatin structure. Chromatin remodelers, among others the INO80 complex, help organize chromatin by repositioning, reshaping, or evicting nucleosomes. We report on INO80 nucleosome remodeling, assayed by single-molecule Foerster resonance energy transfer on canonical nucleosomes as well as nucleosomes assembled from tailless histones. Nucleosome repositioning by INO80 is a processively catalyzed reaction. During the initiation of remodeling, probed by the INO80 bound state, the nucleosome reveals structurally heterogeneous states for tailless nucleosomes (in contrast to wild-type nucleosomes). We, therefore, propose an altered energy landscape for the INO80-mediated nucleosome sliding reaction in the absence of histone tails.

The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling.

Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180-kDa Arp8 module of Saccharomyces cerevisiae INO80 and establish its role in recognition of extranucleosomal linker DNA. Arp8 engages N-actin in a manner distinct from that of other actin-fold proteins and thereby specifies recruitment of the Arp4-N-actin heterodimer to a segmented scaffold of the helicase-SANT-associated (HSA) domain of Ino80. The helical HSA domain spans over 120 Å and provides an extended binding platform for extranucleosomal entry DNA that is required for nucleosome sliding and genome-wide nucleosome positioning. Together with the recent cryo-electron microscopy structure of INO80Core-nucleosome complex, our findings suggest an allosteric mechanism by which INO80 senses 40-bp linker DNA to conduct highly processive chromatin remodeling.