Anders Virtanen

A 54-kDa Fragment of the Poly(A)-specific Ribonuclease Is an Oligomeric, Processive, and Cap-interacting Poly(A)-specific 3′ Exonuclease

We have previously identified a HeLa cell 3′ exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3′ exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180–220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5′-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3′ end-located poly(A) was degraded by the activity. RNA substrates capped with m7G(5′)ppp(5′)G were more efficiently degraded than noncapped RNA substrates. Addition of free m7G(5′)ppp(5′)G cap analogue inhibited poly(A) degradationin vitro, suggesting a functional link between the RNA 5′ end cap structure and poly(A) degradation at the 3′ end of the RNA.

In vitro deadenylation of mammalian mRNA by a HeLa cell 3' exonuclease.

We have identified a 3′ exonuclease in HeLa cell extracts which deadenylates mammalian mRNA and leaves the mRNA body intact after poly(A) removal. Only homopolymeric adenosine tails located at the 3′ end were efficiently removed by the exonuclease. The poly(A) removing activity did not require any specific sequences in the mRNA body either for poly(A) removal or for accumulation of the deadenylated mRNA. We conclude that the poly(A) removing activity is a 3′ exonuclease since (i) reaction intermediates gradually lose the poly(A) tail, (ii) degradation is prevented by the presence of a cordycepin residue at the 3′ end and (iii) RNAs having internally located poly(A) stretches are poor substrates for degradation. The possible involvement of the poly(A) removing enzyme in regulating mRNA translation and stability is discussed.

Nuclear deadenylation/polyadenylation factors regulate 3 ' processing in response to DNA damage

We previously showed that mRNA 3′ end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA‐damaging conditions. The cleavage stimulation factor‐50 (CstF‐50) has a role in this response, providing a link between transcription‐coupled RNA processing and DNA repair. In this study, we show that CstF‐50 interacts with nuclear poly(A)‐specific ribonuclease (PARN) using in vitro and in extracts of UV‐exposed cells. The CstF‐50/PARN complex formation has a role in the inhibition of 3′ cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV‐induced inhibition of 3′ cleavage, strongly activates deadenylation by PARN in the presence of CstF‐50, and that CstF‐50/BARD1 can revert the cap‐binding protein‐80 (CBP80)‐mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA‐damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour‐suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.

Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC).

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3′-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.

Bone marrow failure and developmental delay caused by mutations in poly(A)-specific ribonuclease (PARN)

Background Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. Methods We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. Results We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. Conclusions Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.

Identification of the Major Spliceosomal RNAs in Dictyostelium discoideum Reveals Developmentally Regulated U2 Variants and Polyadenylated snRNAs

ABSTRACT Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5′ cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5′ end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.

Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.

A multifunctional RNA recognition motif in poly(A)-specific ribonuclease with cap and poly(A) binding properties

Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3′ exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.

Poly(A)-specific ribonuclease (PARN): An allosterically regulated, processive and mRNA cap-interacting deadenylase

Abstract Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m7G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3′–5′ exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.

Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a mammalian 3′-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), ∼10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.