MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. In order to silence many different mRNAs in a precise manner, miRNA stability and efficacy is controlled by highly developed regulatory pathways and fine-tuning mechanisms both affecting miRNA processing and altering mature miRNA target specificity.

Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.

Cytoplasmic terminal uridylyltransferases (TUTases) comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor suppressor let-7 miRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe TUTase Cid1 in two Apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP. We also describe a novel high-affinity RNA substrate binding mechanism of Cid1, which is essential for its enzymatic activity and is mediated by three basic patches across the surface of the enzyme. Overall, our structures provide a basis for understanding the activity of Cid1 and a mechanism of UTP selectivity conserved in its human orthologs, with potential implications for anti-cancer drug design.The post-transcriptional addition of uridyl ribonucleotides to cytoplasmic RNA 3′ ends has recently been implicated in several key aspects of eukaryotic RNA biology, including mRNA turnover and regulation of the biogenesis and activity of microRNAs (miRNAs) [1][2][3][4][5][6][7] . In let-7 tumour suppressor miRNA biogenesis, the cytoplasmic terminal U-transferase (TUTase) ZCCHC11 (also known as TUT4 or PUP-2) catalyses the 3′ uridylation of cytoplasmic let-7 precursors (pre-miRNAs), which targets them for destruction 1,2,4 . Additionally, ZCCHC11-dependent uridylylation is important in the regulation of mature miRNAs 3 and replication-dependent histone mRNAs in human cells 6 . In the fission yeast Accession codesThe structures reported in this manuscript have all been deposited in the RCSB PDB, accession codes 4e7x (crystal form I, Apo I conformer), 4e80 (UTP-bound) and 4e8f (crystal form II containing Apo II conformer). Schizosaccharomyces pombe the orthologous enzyme, Cid1 (caffeine-induced death suppressor protein 1), uridylylates polyadenylated mRNAs and stimulates their decay 8,9 . These enzymes belong to the same family as the nuclear poly(A) polymerase 10,11 , but the structural basis for their RNA binding and UTP selectivity has not yet been described. Europe PMC Funders GroupCid1 is a 46 kDa protein containing two recognisable sequence motifs: a nucleotidyl transferase motif common to all members of the DNA polymerase β (Polβ) superfamily and a poly(A) polymerase (PAP)-associated motif. By contrast, ZCCHC11 is much larger (185 kDa), containing a duplication of both motifs found in Cid1, as well as three CCHC-type zinc knuckle motifs ( Supplementary Fig. 1). The C-terminal Cid1-homologous region in ZCCHC11 is catalytically active 3 and shows striking domain conservation ( Supplementary Fig. 1b and c). ZCCHC11 interacts with the RNA-binding protein Lin28 in order to associate stably with (and uridylylate) pre-miRNAs of the let-7 family 1,2,4 and ZCCHC11 inhibition in Lin28A-expressing cancer cells resulted in tumor regression and suppression of invasiveness 12 . No equivalent RNA-binding partner has been descr...

SummaryEfficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

Homologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase. Keywords Homologous recombination • Cryo electron microscopy • X-ray crystallography • Double-strand break repair • DNA damage signalling and repair Abbreviations ADP Adenosine diphosphate AMP-PNP Adenylyl-imidodiphosphate ATM Ataxia telangiectasia mutated ATP Adenosine triphosphate ATR Ataxia telangiectasia and Rad3-related protein BARD1 BRCA1-associated RING domain protein 1 BRCA1 Breast cancer type 1 susceptibility protein BRCA2 Breast cancer type 2 susceptibility protein BRCT BRCA1 C-terminal domain cryoEM Cryo electron microscopy DDR DNA damage response DSB Double-strand break EJ End joining MRE11 Meiotic recombination 11 homolog 1 MRN MRE11 RAD50 NBS1 NBS1 Nijmegen breakage syndrome protein 1 OB Oligonucleotide/oligosaccharide-binding PALB2 Partner and localiser of BRCA2 RPA Replication protein A

Highlights d cryo-EM structure of Tel1 in complex with Mg 2+-AMP-PNP at 3.7 Å resolution d Key residues of the active site are in productive conformation for catalysis d PIKK regulatory domain insert (PRD-I) restricts peptide substrate access d Structural rational for mutations found in ataxiatelangiectasia and cancer

In response to DNA damage or replication fork stalling, the basal activity of Saccharomyces cerevisiae Mec1 ATR is stimulated in a cell cycle dependent manner, leading to cell cycle arrest and promoting DNA repair. Mec1 ATR dysfunction leads to cell death in yeast and causes chromosome instability and embryonic lethality in mammals. Thus, ATR is a major target for cancer therapies in homologous recombination-deficient cancers. Here we identify a single mutation in Mec1, conserved in ATR, that results in constitutive activity. Using cryo electron microscopy, we determined the structures of this constitutively active form (Mec1(F2244L)-Ddc2) at 2.8 Å, and the wild-type at 3.8 Å, both in complex with Mg 2+ -AMP-PNP. These structures yield near complete atomic models for Mec1-Ddc2 and uncover the molecular basis for low basal activity and the conformational changes required for activation. Combined with biochemical and genetic data, we discover key regulatory regions and propose a Mec1 activation mechanism.

Background: Kindlins are essential co-activators of integrins.Results: Kindlin-3 has an elongated structure and forms a ternary complex with the Talin head and integrin β-tails. The Kindlin-3-tail interface involves a membrane-distal NPXY motif on the tail.Conclusion: New information about the conformation and interactions of Kindlin-3 has been obtained.Significance: The solution structure and protein/protein interactions of Kindlin-3 give insight into its role.