Exonuclease hDIS3L2 specifies an exosome‐independent 3′‐5′ degradation pathway of human cytoplasmic mRNA

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5′‐3′ exoribonuclease hXRN1 and the 3′‐5′ exoribonucleolytic RNA exosome complex. However, in addition to the exosome‐associated 3′‐5′ exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein—hDIS3L2. Here, we show that hDIS3L2 is an exosome‐independent cytoplasmic mRNA 3′‐5′ exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA‐dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P‐bodies) upon hDIS3L2 depletion, which also increases half‐lives of investigated mRNAs. Consistently, RNA sequencing (RNA‐seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome‐independent effector of cytoplasmic mRNA metabolism.     

A protein complex mediating mRNA degradation in Escherichia coli

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribolucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease poly-nucleotide phosphorylase (PNPase) and the endorlbo-nuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional poly-peptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.     

Excision of β-L- and β-D-Nucleotide Analogs From DNA by p53 Protein

Abstract

The tumor suppressor p₅₃ protein plays a critical role in the cell-cycle progression. The role of the 3′-to-5′ exonuclease activity of p53 protein in the DNA repair process remains elusive. Using an in vitro exonuclease assay and defined oligonucleotides terminated with β-D- and β-L-nucleoside analogs at the 3′-terminus, we studied the ability of p53 protein to excise β-L- and β-D-nucleoside analogs which have anticancer or antiviral potential. p53 protein removes β-D-nucleoside analogs more efficiently compared to that of β-L-nucleoside analogs. The affinity of p53 protein for an β-L-nucleotide terminated primer was 5 fold lower compared to non-modified primer. The hypothesis on an important role of the 3′-to-5′ exonuclease activity of p53 protein in the action of nucleoside analogs was proposed.     

Synthetic repetitive extragenic palindromic (REP) sequence as an efficient mRNA stabilizer for protein production and metabolic engineering in prokaryotic cells

In prokaryotic cells, 3′–5′ exonucleases can attenuate messenger RNA (mRNA) directionally from the direction of the 3′–5′ untranslated region (UTR), and thus improving the stability of mRNAs without influencing normal cell growth and metabolism is a key challenge for protein production and metabolic engineering. Herein, we significantly improved mRNA stability by using synthetic repetitive extragenic palindromic (REP) sequences as an effective mRNA stabilizer in two typical prokaryotic microbes, namely, Escherichia coli for the production of cyclodextrin glucosyltransferase (CGTase) and Corynebacterium glutamicum for the production of N-acetylglucosamine (GlcNAc). First, we performed a high-throughput screen to select 4 out of 380 REP sequences generated by randomizing 6 nonconservative bases in the REP sequence designed as the degenerate base “N.” Secondly, the REP sequence was inserted at several different positions after the stop codon of the CGTase-encoding gene. We found that mRNA stability was improved only when the space between the REP sequence and stop codon was longer than 12 base pairs (bp). Then, by reconstructing the spacer sequence and secondary structure of the REP sequence, a REP sequence with 8 bp in a stem-loop was obtained, and the CGTase activity increased from 210.6 to 291.5 U/ml. Furthermore, when this REP sequence was added to the 3′-UTR of glucosamine-6-phosphate N-acetyltransferase 1 ( GNA1), which is a gene encoding a key enzyme GNA1 in the GlcNAc synthesis pathway, the GNA1 activity was increased from 524.8 to 890.7 U/mg, and the GlcNAc titer was increased from 4.1 to 6.0 g/L in C. glutamicum. These findings suggest that the REP sequence plays an important function as an mRNA stabilizer in prokaryotic cells to stabilize its 3′-terminus of the mRNA by blocking the processing action of the 3′–5′ exonuclease. Overall, this study provides new insight for the high-efficiency overexpression of target genes and pathway fine-tuning in bacteria.     

Function of 3′ non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3 untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3 flanking sequences of psaB or extended the open reading frame into the 3 inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3 stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3 inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.     

Studies of the 5' exonuclease and endonuclease activities of CPSF-73 in histone pre-mRNA processing

Processing of histone pre-mRNA requires a single 3′ endonucleolytic cleavage guided by the U7 snRNP that binds downstream of the cleavage site. Following cleavage, the downstream cleavage product (DCP) is rapidly degraded in vitro by a nuclease that also depends on the U7 snRNP. Our previous studies demonstrated that the endonucleolytic cleavage is catalyzed by the cleavage/polyadenylation factor CPSF-73. Here, by using RNA substrates with different nucleotide modifications, we characterize the activity that degrades the DCP. We show that the degradation is blocked by a 2′-O-methyl nucleotide and occurs in the 5′-to-3′ direction. The U7-dependent 5′ exonuclease activity is processive and continues degrading the DCP substrate even after complete removal of the U7-binding site. Thus, U7 snRNP is required only to initiate the degradation. UV cross-linking studies demonstrate that the DCP and its 5′-truncated version specifically interact with CPSF-73, strongly suggesting that in vitro, the same protein is responsible for the endonucleolytic cleavage of histone pre-mRNA and the subsequent degradation of the DCP. By using various RNA substrates, we define important space requirements upstream and downstream of the cleavage site that dictate whether CPSF-73 functions as an endonuclease or a 5′ exonuclease. RNA interference experiments with HeLa cells indicate that degradation of the DCP does not depend on the Xrn2 5′ exonuclease, suggesting that CPSF-73 degrades the DCP both in vitro and in vivo.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.     

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.     

DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II

The Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single-stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.     

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

RNase J1, a ribonuclease with 5′ exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5′ end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3′ exonucleases; the downstream fragment is degraded by RNase J1 5′ exonuclease activity. Previously, ΔermC mRNA was used to show 5′-end dependence of mRNA turnover. Here we used ΔermC mRNA to probe RNase J1-dependent degradation, and the results were consistent with aspects of the model. ΔermC mRNA showed increased stability in a mutant strain that contained a reduced level of RNase J1. In agreement with the tracking concept, insertion of a strong stem–loop structure at +65 resulted in increased stability. Weakening this stem–loop structure resulted in reversion to wild-type stability. RNA fragments containing the 3′ end were detected in a strain with reduced RNase J1 expression, but were undetectable in the wild type. The 5′ ends of these fragments mapped to the upstream side of predicted stem–loop structures, consistent with an impediment to RNase J1 5′ exonuclease processivity. A ΔermC mRNA deletion analysis suggested that decay-initiating endonuclease cleavage could occur at several sites near the 3′ end. However, even in the absence of these sites, stability was further increased in a strain with reduced RNase J1, suggesting alternate pathways for decay that could include exonucleolytic decay from the 5′ end.     

Messenger RNA Turnover in Eukaryotes: Pathways and Enzymes

The control of mRNA degradation is an important component of the regulation of gene expression since the steady-state concentration of mRNA is determined both by the rates of synthesis and of decay. Two general pathways of mRNA decay have been described in eukaryotes. Both pathways share the exonucleolytic removal of the poly(A) tail (deadenylation) as the first step. In one pathway, deadenylation is followed by the hydrolysis of the cap and processive degradation of the mRNA body by a 5′ exonuclease. In the second pathway, the mRNA body is degraded by a complex of 3′ exonucleases before the remaining cap structure is hydrolyzed. This review discusses the proteins involved in the catalysis and control of both decay pathways.     

Purification and characterization of exonuclease-free Artemis: Implications for DNA-PK-dependent processing of DNA termini in NHEJ-catalyzed DSB repair

Artemis is a member of the β-CASP family of nucleases in the metallo-β-lactamase superfamily of hydrolases. Artemis has been demonstrated to be involved in V(D)J-recombination and in the NHEJ-catalyzed repair of DNA DSBs. In vitro, both DNA-PK independent 5′–3′ exonuclease activities and DNA-PK dependent endonuclease activity have been attributed to Artemis, though mutational analysis of the Artemis active site only disrupts endonuclease activity. This suggests that either the enzyme contains two different active sites, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. Recombinant [His]₆-Artemis was expressed in a Baculovirus insect-cell expression system and isolated using a three-column purification methodology. Exonuclease and endonuclease activities, the ability to be phosphorylated by DNA-PK, and Artemis antibody reactivity was monitored throughout the purification and to characterize final pools of protein preparation. Results demonstrated the co-elution of exonuclease and endonuclease activities on a Ni–agarose affinity column but separation of the two enzymatic activities upon fractionation on a hydroxyapatite column. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease.     

Blocking polyadenylation of mRNA in the chloroplast inhibits its degradation

The addition of poly(A)-rich sequences to endonuclease cleavage products of chloroplast mRNA has recently been suggested to target the polyadenylated RNA for rapid exonucleolytic degradation. This study analyzed whether the addition of a poly(A)-rich tail to RNA molecules is required for degradation by chloroplast exonuclease(s). In lyzed chloroplasts from spinach, addition of the polyadenylation inhibitor, cordycepin triphosphate (3′-dATP), inhibited the degradation of psbA and rbcL mRNAs. Furthermore, degradation intermediates generated by endonucleolytic cleavages accumulated. Similar results were obtained when yeast tRNA was added to the mRNA degradation system as a non-specific exoribonuclease inhibitor. Nevertheless, the stabilization mechanisms differ: while tRNA directly affects the exonuclease activity, 3′dATP has an indirect effect by inhibiting polyadenylation. The results indicate that the addition of poly(A)-rich sequences to endonucleolytic cleavage products of chloroplast mRNA is required to target these RNAs for rapid exonucleolytic degradation. Together with previous work, the data reported here support a model for mRNA degradation in the chloroplast in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the proximal cleavage products, targeting these RNAs for rapid exonucleolytic decay.     

Tracking the elusive 5′ exonuclease activity of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> RNase J

Key message

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.

Abstract

RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.