A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in viv

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs—the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein function

Structural insights into the 3′-end mRNA maturation machinery: Snapshot on polyadenylation signal recognition

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Complex behavior: from cannibalism to suicide in the vitamin B1 biosynthesis world

Although thiamin was the first vitamin discovered over a century ago, it is only within the last decade that its metabolism has begun to be unraveled. Over the last few years, several structural and biochemical studies have provided insight into the unprecedented mechanisms of the proteins involved, revealing some remarkable biochemistry. Thiamin biosynthesis is particularly unusual in eukaryotes (fungi and plants) in that it cannibalizes essential cellular cofactors and relies on single turnover proteins, which succumb to enzymatic suicide. Here we provide an overview of recent structural studies that have advanced our understanding of this vital metabolite and question whether the single turnover proteins act to monitor the level of the essential elements used as substrates

Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand

Riboswitches are untranslated regions of messenger RNA, which adopt alternate structures depending on the binding of specific metabolites. Such conformational switching regulates the expression of proteins involved in the biosynthesis of riboswitch substrates. Here, we present the 2.9 angstrom-resolution crystal structure of the eukaryotic Arabidopsis thaliana thiamine pyrophosphate (TPP)-specific riboswitch in complex with its natural ligand. The riboswitch specifically recognizes the TPP via conserved residues located within two highly distorted parallel "sensor" helices. The structure provides the basis for understanding the reorganization of the riboswitch fold upon TPP binding and explains the mechanism of resistance to the antibiotic pyrithiamine

Domain definition and interaction mapping for the endonuclease complex hNob1/hPno1: endonuclease complex hNob1/hPno1 interaction

International audienceRibosome biogenesis requires a variety of trans-acting factors in order to produce functional ribosomal subunits. In human cells, the complex formed by the proteins hNob1 and hPno1 is crucial to the site 3 cleavage occurring at the 3'-end of 18S pre-rRNA. However, the properties and activity of this complex are still poorly understood. We present here a detailed characterization of hNob1 organization and its interaction with hPno1. We redefine the boundaries of the endonuclease PIN domain present in hNob1 and we further delineate the precise interacting modules required for complex formation in hNob1 and hPno1. Altogether, our data contributes to a better understanding of the complex biology required during the site 3 cleavage step in ribosome biogenesis

Structural basis of thiamine pyrophosphate analogues binding to the eukaryotic riboswitch

The thiamine pyrophosphate (TPP)-sensing riboswitch is the only riboswitch found in eukaryotes. In plants, TPP regulates its own production by binding to the 3' untranslated region of the mRNA encoding ThiC, a critical enzyme in thiamine biosynthesis, which promotes the formation of an unstable splicing variant. In order to better understand the molecular basis of TPP-analogue binding to the eukaryotic TPP-responsive riboswitch, we have determined the crystal structures of the Arabidopsis thaliana TPP-riboswitch in complex with oxythiamine pyrophosphate (OTPP) and with the antimicrobial compound pyrithiamine pyrophosphate (PTPP). The OTPP-riboswitch complex reveals that the pyrimidine ring of OTPP is stabilized in its enol form in order to retain key interactions with guanosine 28 of the riboswitch previously observed in the TPP complex. The structure of PTPP in complex with the riboswitch shows that the base moiety of guanosine 60 undergoes a conformational change to cradle the pyridine ring of the PTPP. Structural information from these complexes has implications for the design of novel antimicrobials targeting TPP-sensing riboswitches

Functional implications from the Cid1 poly(U) polymerase crystal structure

In eukaryotes, mRNA degradation begins with poly(A) tail removal, followed by decapping, and the mRNA body is degraded by exonucleases. In recent years, the major influence of 3'-end uridylation as a regulatory step within several RNA degradation pathways has generated significant attention toward the responsible enzymes, which are called poly(U) polymerases (PUPs). We determined the atomic structure of the Cid1 protein, the founding member of the PUP family, in its UTP-bound form, allowing unambiguous positioning of the UTP molecule. Our data also suggest that the RNA substrate accommodation and product translocation by the Cid1 protein rely on local and global movements of the enzyme. Supplemented by point mutations, the atomic model is used to propose a catalytic cycle. Our study underlines the Cid1 RNA binding properties, a feature with critical implications for miRNAs, histone mRNAs, and, more generally, cellular RNA degradation

A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure

The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo

X-ray structure and activity of the yeast Pop2 protein: a nuclease subunit of the mRNA deadenylase complex

In Saccharomyces cerevisiae, a large complex, known as the Ccr4–Not complex, containing two nucleases, is responsible for mRNA deadenylation. One of these nucleases is called Pop2 and has been identified by similarity with PARN, a human poly(A) nuclease. Here, we present the crystal structure of the nuclease domain of Pop2 at 2.3 Å resolution. The domain has the fold of the DnaQ family and represents the first structure of an RNase from the DEDD superfamily. Despite the presence of two non-canonical residues in the active site, the domain displays RNase activity on a broad range of RNA substrates. Site-directed mutagenesis of active-site residues demonstrates the intrinsic ability of the Pop2 RNase D domain to digest RNA. This first structure of a nuclease involved in the 3′–5′ deadenylation of mRNA in yeast provides information for the understanding of the mechanism by which the Ccr4–Not complex achieves its functions