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...