Megerditch Kiledjian

MicroRNAs (miRNAs) are endogenous single-stranded RNA molecules of about 21 nucleotides in length that are fundamental post-transcriptional regulators of gene expression. Although the transcriptional and processing events involved in the generation of miRNAs have been extensively studied, very little is known pertaining to components that regulate the stability of individual miRNAs. All RNAs have distinct inherent half-lives that dictate their level of accumulation and miRNAs would be expected to follow a similar principle. Here we demonstrate that although most miRNA appear to be stable, like mRNAs, miRNAs possess differential stability in human cells. In particular, we found that miR-382, a miRNA that contributes to HIV-1 provirus latency, is unstable in cells. To determine the region of miR-382 responsible for its rapid decay, we developed a cell-free system that recapitulated the observed cell-based-regulated miR-382 turnover. The system utilizes in vitro-processed mature miRNA derived from pre-miRNA and follows the decay of the processed miRNA. Using this system, we demonstrate that instability of miR-382 is driven by sequences outside its seed region and required the 3′ terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 3′–5′ exoribonuclease complex was identified as the primary nuclease involved in miR-382 decay with a more modest contribution by the Xrn1 and no detectable contribution by Xrn2. These studies provide evidence for an miRNA element essential for rapid miRNA decay and implicate the exosome in this process. The development of a biochemically amendable system to analyze the mechanism of differential miRNA stability provides an important step in efforts to regulate gene expression by modulating miRNA stability.

Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5′ cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m⁷GpppN. HIT proteins are homodimeric and contain two conserved 100-aminoacid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxylterminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.

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Regulation of decapping is a critical determinant of mRNA stability. We recently identified hDcp2 as a human decapping enzyme with intrinsic decapping activity. This activity is specific to N⁷-methylated guanosine containing RNA. The hDcp2 enzyme does not function on the cap structure alone and is not sensitive to competition by cap analog, suggesting that hDcp2 requires the RNA for cap recognition. We now demonstrate that hDcp2 is an RNA-binding protein and its recognition and hydrolysis of the cap substrate is dependent on an initial interaction with the RNA moiety. A biochemical characterization of hDcp2 revealed that a 163 amino acid region containing two evolutionarily conserved regions, the Nudix fold hydrolase domain and the adjacent Box B region contained methyl-cap-specific hydrolysis activity. Maximum decapping activity for wild-type as well as truncation mutants of hDcp2 required Mn²⁺as a divalent cation. The demonstration that hDcp2 is an RNA-binding protein with an RNA-dependent decapping activity will now provide new approaches to identify specific mRNAs that are regulated by this decapping enzyme as well as provide novel avenues to control mRNA decapping and turnover by influencing the RNA-binding property of hDcp2.

Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals. We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity. hDcp2 specifically hydrolyzed methylated capped RNA to release m ⁷ GDP; however, it did not function on the cap structure alone. hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS. hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity. hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes. Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells. Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping. Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity.

We recently demonstrated that the major decapping activity in mammalian cells involves DcpS, a scavenger pyrophosphatase that hydrolyzes the residual cap structure following 3′ to 5′ decay of an mRNA. The association of DcpS with 3′ to 5′ exonuclease exosome components suggests that these two activities are linked and there is a coupled exonucleolytic decay‐dependent decapping pathway. We purified DcpS from mammalian cells and identified the cDNA encoding a novel 40 kDa protein possessing DcpS activity. Consistent with purified DcpS, the recombinant protein specifically hydrolyzed methylated cap analog but did not hydrolyze unmethylated cap analog nor did it function on intact capped RNA. Sequence alignments of DcpS from different organisms revealed the presence of a conserved hexapeptide, containing a histidine triad (HIT) sequence with three histidines separated by hydrophobic residues. Mutagenesis analysis revealed that the central histidine within the DcpS HIT motif is critical for decapping activity and defines the HIT motif as a new mRNA decapping domain, making DcpS the first member of the HIT family of proteins with a defined biological function.

The α-globin mRNA contains a C-rich stability element (CRE) in its 3′ untranslated region (3′ UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the α-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the α-globin 3′ UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the α-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of α-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the α-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3′ to 5′ exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the α-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3′-to-5′ decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.

The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozy

Stability of the human α‐globin mRNA is conferred by a ribonucleoprotein complex termed the α‐complex, which acts by impeding deadenylation. Using our recently devised in vitro decay assay, we demonstrate that the α‐complex also functions by protecting the 3′‐untranslated region (3′‐UTR) from an erythroid‐enriched, sequence‐specific endoribonuclease activity. The cleavage site was mapped to a region protected by the α‐complex and is regulated by the presence of the α‐complex. Similar endoribonuclease cleavage products were also detected in erythroid cells expressing an exogenous α‐globin gene. Nucleotide substitution of the target sequence renders the RNA refractory to the endoribonuclease activity. Insertion of the target sequence onto a heterologous RNA confers sequence‐specific cleavage on the chimeric RNA, demonstrating the sequence specificity of this activity. We conclude that the α‐complex stabilizes the α‐globin mRNA in erythroid cells by a multifaceted approach, one aspect of which is to protect the 3′‐UTR from specific endoribonuclease cleavage.

Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and αCP1, a protein implicated in α-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein–RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by αCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3′ UTRs consistent with the binding of αCP1. Furthermore, direct binding of purified αCP proteins to these 3′ UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions.