Megerditch Kiledjian

The hDcp2 protein is a mammalian mRNA decapping enzyme.

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 m7GDP; 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.

An mRNA Stability Complex Functions with Poly(A)-Binding Protein To Stabilize mRNA In Vitro

ABSTRACT The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. α-Globin mRNA stability is dictated by sequences in the 3′ untranslated region (3′UTR) which form a specific ribonucleoprotein complex (α-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, αCP1 and αCP2. Using an in vitro-transcribed and polyadenylated α-globin 3′UTR, we have devised an in vitro mRNA decay assay which reproduces the α-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the α-complex by sequestration of αCP1 and αCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the α-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between αCP1 and αCP2 with PABP suggests that the α-complex can directly interact with PABP. Therefore, the α-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases

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.

Reversible methylation of m6Am in the 5′ cap controls mRNA stability

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Structure and function of the 5'→3' exoribonuclease Rat1 and its activating partner Rai1

The 5′→3′ exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7–14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 Å resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 Å resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5′ triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.

Identification of a quality-control mechanism for mRNA 5′-end capping

The 7-methylguanosine cap structure at the 5′ end of eukaryotic messenger RNAs is a critical determinant of their stability and translational efficiency. It is generally believed that 5′-end capping is a constitutive process that occurs during mRNA maturation and lacks the need for a quality-control mechanism to ensure its fidelity. We recently reported that the yeast Rai1 protein has pyrophosphohydrolase activity towards mRNAs lacking a 5′-end cap. Here we show that, in vitro as well as in yeast cells, Rai1 possesses a novel decapping endonuclease activity that can also remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Capped but unmethylated mRNAs generated in yeast cells with a defect in the methyltransferase gene are more stable in a rai1-gene-disrupted background. Moreover, rai1Δ yeast cells with wild-type capping enzymes show significant accumulation of mRNAs with 5′-end capping defects under nutritional stress conditions of glucose starvation or amino acid starvation. These findings provide evidence that 5′-end capping is not a constitutive process that necessarily always proceeds to completion and demonstrates that Rai1 has an essential role in clearing mRNAs with aberrant 5′-end caps. We propose that Rai1 is involved in an as yet uncharacterized quality control process that ensures mRNA 5′-end integrity by an aberrant-cap-mediated mRNA decay mechanism.

The poly(A)-binding protein and an mRNA stability protein jointly regulate an endoribonuclease activity.

ABSTRACT We previously identified a sequence-specific erythroid cell-enriched endoribonuclease (ErEN) activity involved in the turnover of the stable α-globin mRNA. We now demonstrate that ErEN activity is regulated by the poly(A) tail. The unadenylated α-globin 3′ untranslated region (3′UTR) was an efficient substrate for ErEN cleavage, while the polyadenylated 3′UTR was inefficiently cleaved in an in vitro decay assay. The influence of the poly(A) tail was mediated through the poly(A)-binding protein (PABP) bound to the poly(A) tail, which can inhibit ErEN activity. ErEN cleavage of an adenylated α-globin 3′UTR was accentuated upon depletion of PABP from the cytosolic extract, while addition of recombinant PABP reestablished the inhibition of endoribonuclease cleavage. PABP inhibited ErEN activity indirectly through an interaction with the αCP mRNA stability protein. Sequestration of αCP resulted in an increase of ErEN cleavage activity, regardless of the polyadenylation state of the RNA. Using electrophoretic mobility shift assays, PABP was shown to enhance the binding efficiency of αCP to the α-globin 3′UTR, which in turn protected the ErEN target sequence. Conversely, the binding of PABP to the poly(A) tail was also augmented by αCP, implying that a stable higher-order structural network is involved in stabilization of the α-globin mRNA. Upon deadenylation, the interaction of PABP with αCP would be disrupted, rendering the α-globin 3′UTR more susceptible to endoribonuclease cleavage. The data demonstrated a specific role for PABP in protecting the body of an mRNA in addition to demonstrating PABPs well-characterized effect of stabilizing the poly(A) tail.

DcpS as a therapeutic target for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene, which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene,SMN2, which produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by 2-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-substituted quinazoline probe, we identified the scavenger decapping enzyme, DcpS, as a potential binder. We show that the C5-substituted quinazolines potently inhibit DcpS decapping activity and that the potency of inhibition correlates with potency forSMN2 promoter induction. Binding of C5-substituted quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m(7)GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.

Identification of Target Messenger RNA Substrates for the Murine Deleted in Azoospermia-Like RNA-Binding Protein

Abstract The murine autosomal deleted in azoospermia-like protein (mDAZL) is a germ cell-restricted RNA-binding protein essential for sperm production. Homozygous disruption of the mDAZL gene results in the absence of germ cells beyond the spermatogonial stage. Progress into the function of DAZL in spermatogenesis has been hampered without identification of the cognate mRNA substrates that it binds to and regulates. Using the isolation of specific nucleic acids associated with proteins (SNAAP) technique recently developed in our lab, we identified mRNAs from testis that were specifically bound by mDAZL. One mRNA encoded the Tpx-1 protein, a testicular cell adhesion protein essential for the progression of spermatogenesis. A 26-nucleotide region necessary and sufficient to bind mDAZL was found within additional mRNAs isolated by the screen. These included mRNA encoding Pam, a protein associated with myc; GRSF1, an mRNA-binding protein involved in translation activation, and TRF2, a TATA box-binding protein-like protein involved in transcriptional regulation. Each mRNA containing the mDAZL binding site was specifically bound by mDAZL. A similar sequence is also present in the Cdc25A mRNA, a threonine/tyrosine phosphatase involved in cell cycle progression. The mDAZL and Cdc25A homologues are functionally linked in Drosophila and are necessary for spermatogenesis. Our demonstration that Tpx-1 and Cdc25A mRNAs are bound by mDAZL suggests that mDAZL regulates a subset of mRNAs necessary for germ cell development and cell cycle progression. Understanding how mDAZL regulates the target mRNAs will provide new insights into spermatogenesis, strategies for therapeutic intervention in azoospermic patients, and novel approaches for male contraception.

Identification of an erythroid-enriched endoribonuclease activity involved in specific mRNA cleavage

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.