Endonuclease-mediated mRNA decay requires tyrosine phosphorylation of polysomal ribonuclease 1 (PMR1) for the targeting and degradation of polyribosome-bound substrate mRNA

PMR1 is an endonuclease that is activated by estrogen to degrade Xenopus albumin mRNA. A previous report showed that the functional unit of endonuclease-mediated mRNA decay is a approximately 680-kDa polysome-bound complex that contains both PMR1 and substrate mRNA. PMR1 contains two domains involved in endonuclease targeting to polysomes, an N-terminal domain that lies between residues 200 and 250, and a C-terminal domain that lies within the last 100 residues. Loss of either domain inactivated PMR1 targeting to polysomes and stabilized albumin mRNA. The current study identified a phosphorylated tyrosine residue within the C-terminal polysome-targeting domain and showed that this modification is required for PMR1-mediated mRNA decay. Changing this tyrosine to phenylalanine inactivated the targeting of PMR1 to polysomes, blocked binding of PMR1 to the functional complex containing its substrate mRNA, prevented the targeting of a green fluorescent protein fusion protein to this complex, and stabilized albumin mRNA to degradation by PMR1 in vivo. A general tyrosine kinase inhibitor inhibited the phosphorylation of PMR1, which in turn inhibited PMR1-catalyzed degradation of albumin mRNA. These results indicate that one or more tyrosine kinases functions as a regulator of endonuclease-mediated mRNA decay.     

The 90-kDa heat shock protein stabilizes the polysomal ribonuclease 1 mRNA endonuclease to degradation by the 26S proteasome

The polysomal ribonuclease 1 (PMR1) mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and it identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that 90-kDa heat shock protein (Hsp90) is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes.     

c-Src activates endonuclease-mediated mRNA decay

The mRNA endonuclease PMR1 initiates mRNA decay by forming a selective complex with its translating substrate mRNA. Previous work showed that the ability of PMR1 to target to polysomes and activate decay depends on the phosphorylation of a tyrosine residue at position 650. The current study shows that c-Src is responsible for activating this mRNA decay pathway. c-Src was recovered with immunoprecipitated PMR1, and it phosphorylates PMR1 in vitro and in vivo. The interaction with c-Src involves two domains of PMR1: Y650 and a series of proline-rich SH3 peptides in the N terminus. In cells with little c-Src, PMR1 targeting to polysomes is induced by constitutively active c-Src but not by inactive forms of the kinase. Similarly, only active c-Src induces PMR1-mediated mRNA decay. Finally, we show that EGF rapidly induces c-Src phosphorylation of PMR1, providing a direct link between tyrosine kinase-mediated signal transduction and mRNA decay.     

Polysome-bound endonuclease PMR1 is targeted to stress granules via stress-specific binding to TIA-1

The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional approximately 680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.     

An endonuclease activity similar to Xenopus PMR1 catalyzes the degradation of normal and nonsense-containing human beta-globin mRNA in erythroid cells

beta-globin mRNA bearing a nonsense codon is degraded in the cytoplasm of erythroid cells by endonuclease cleavage, preferentially at UG dinucleotides. An endonuclease activity in polysomes of MEL cells cleaved beta-globin and albumin mRNA in vitro at many of the same sites as PMR1, an mRNA endonuclease purified from Xenopus liver. Stable transfection of MEL cells expressing normal human beta-globin mRNA with a plasmid vector expressing the catalytically active form of PMR1 reduced the half-life of beta-globin mRNA from 12 to 1-2 h without altering GAPDH mRNA decay. The reduced stability of beta-globin mRNA in these cells was accompanied by an increase in the production of mRNA decay products corresponding to those seen in the degradation of nonsense-containing beta-globin mRNA. Therefore, beta-globin mRNA is cleaved in vivo by an endonuclease with properties similar to PMR1. Inhibiting translation with cycloheximide stabilized nonsense-containing beta-globin mRNA, resulting in a fivefold increase in its steady-state level. Taken together, our results indicate that the surveillance of nonsense-containing beta-globin mRNA in erythroid cells is a cytoplasmic process that functions on translating mRNA, and endonucleolytic cleavage constitutes one step in the process of beta-globin mRNA decay.     

A polysomal ribonuclease involved in the destabilization of albumin mRNA is a novel member of the peroxidase gene family.

We have purified an approximately 60 kDa endoribonuclease from Xenopus liver polysomes with properties expected for a messenger RNase involved in the estrogen-regulated destabilization of serum protein mRNAs (Dompenciel et al., 1995, J Biol Chem 270:6108-6118). The present report describes the cloning of this protein and its identification as a novel member of the peroxidase gene family. This novel enzyme, named polysomal RNase 1, or PMR-1 has 57% sequence identity with myeloperoxidase, and like that protein, appears to be processed from a larger precursor. Unlike myeloperoxidase, however, PMR-1 lacks N-linked oligosaccharide, heme, and peroxidase activity. Western blot and immunoprecipitation experiments using epitope-specific antibodies to the derived protein sequence confirm the identity of the cloned cDNA to the protein originally isolated from polysomes. The 80 kDa pre-PMR-1 expressed in a recombinant baculovirus was not processed to the 60 kDa form in Sf9 cells and lacks RNase activity. However, the baculovirus-expressed mature 60-kDa form of the enzyme has RNase activity. The recombinant protein is an endonuclease that shows selectivity for albumin versus ferritin mRNA. While it does not cleave at consensus APyrUGA elements, recombinant PMR-1 generates the same minor cleavage products from albumin mRNA as PMR-1 purified from liver. Finally, we show estrogen induces only a small increase in the amount of PMR-1. This result is consistent with earlier data suggesting estrogen activates mRNA decay through a posttranslational pathway.     

Regulation of c-myc mRNA stability in vitro by a labile destabilizer with an essential nucleic acid component.

The turnover rates of some mRNAs vary by an order of magnitude or more when cells change their growth pattern or differentiate. To identify regulatory factors that might be responsible for this variability, we investigated how cytosolic fractions affect mRNA decay in an in vitro system. A 130,000 X g supernatant (S130) from the cytosol of exponentially growing erythroleukemia cells contains a destabilizer that accelerates the decay of polysome-bound c-myc mRNA by eightfold or more compared with reactions lacking S130. The destabilizer is deficient in or absent from the S130 of cycloheximide-treated cells, indicating that it is labile or is repressed when translation is blocked. It is not a generic RNase, because it does not affect the turnover of delta-globin, gamma-globin, or histone mRNA and does not destabilize a major portion of polysomal polyadenylated mRNA. The destabilizer accelerates the turnover of the c-myc mRNA 3' region, as well as subsequent 3'-to-5' degradation of the mRNA body. It is inactivated in vitro by mild heating and by micrococcal nuclease, suggesting that it contains a nucleic acid component. c-myb mRNA is also destabilized in S130-supplemented in vitro reactions. These results imply that the stability of some mRNAs is regulated by cytosolic factors that are not associated with polysomes.     

Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs.     

Human La protein: a stabilizer of histone mRNA.

Histone mRNA is destabilized at the end of S phase and in cell-free mRNA decay reaction mixtures supplemented with histone proteins, indicating that histones might autoregulate the histone mRNA half-life. Histone mRNA destabilization in vitro requires three components: polysomes, histones, and postpolysomal supernatant (S130). Polysomes are the source of the mRNA and mRNA-degrading enzymes. To investigate the role of the S130 in autoregulation, crude S130 was fractionated by histone-agarose affinity chromatography. Two separate activities affecting the histone mRNA half-life were detected. The histone-agarose-bound fraction contained a histone mRNA destabilizer that was activated by histone proteins; the unbound fraction contained a histone mRNA stabilizer. Further chromatographic fractionation of unbound material revealed only a single protein stabilizer, which was purified to homogeneity, partially sequenced, and found to be La, a well-characterized RNA-binding protein. When purified La was added to reaction mixtures containing polysomes, a histone mRNA decay intermediate was stabilized. This intermediate corresponded to histone mRNA lacking 12 nucleotides from its 3' end and containing an intact coding region. Anti-La antibody blocked the stabilization effect. La had little or no effect on several other cell cycle-regulated mRNAs. We suggest that La prolongs the histone mRNA half-life during S phase and thereby increases histone protein production.     

Nutrients and the Pkh1/2 and Pkc1 protein kinases control mRNA decay and P-body assembly in yeast

Regulated mRNA decay is essential for eukaryotic survival but the mechanisms for regulating global decay and coordinating it with growth, nutrient, and environmental cues are not known. Here we show that a signal transduction pathway containing the Pkh1/Pkh2 protein kinases and one of their effector kinases, Pkc1, is required for and regulates global mRNA decay at the deadenylation step in Saccharomyces cerevisiae. Additionally, many stresses disrupt protein synthesis and release mRNAs from polysomes for incorporation into P-bodies for degradation or storage. We find that the Pkh1/2-Pkc1 pathway is also required for stress-induced P-body assembly. Control of mRNA decay and P-body assembly by the Pkh-Pkc1 pathway only occurs in nutrient-poor medium, suggesting a novel role for these processes in evolution. Our identification of a signaling pathway for regulating global mRNA decay and P-body assembly provides a means to coordinate mRNA decay with other cellular processes essential for growth and long-term survival. Mammals may use similar regulatory mechanisms because components of the decay apparatus and signaling pathways are conserved.     

Identification of in vivo mRNA decay intermediates corresponding to sites of in vitro cleavage by polysomal ribonuclease 1

Previous work from this laboratory identified a polysome-associated endonuclease whose activation by estrogen correlates with the coordinate destabilization of serum protein mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a novel member of the peroxidase gene family. A characteristic feature of PMR-1 is its ability to generate in vitro degradation intermediates by cleaving within overlapping APyrUGA elements in the 5'-coding region of albumin mRNA. The current study sought to determine whether the in vivo destabilization of albumin mRNA following estrogen administration involves the generation of decay intermediates that could be identified as products of PMR-1 cleavage. A sensitive ligation-mediated polymerase chain reaction technique was developed to identify labile decay intermediates, and its validity in identifying PMR-1-generated decay intermediates of albumin mRNA was confirmed by primer extension experiments performed with liver RNA that was isolated from estrogen-treated frogs or digested in vitro with the purified endonuclease. Ligation-mediated polymerase chain reaction was also used to identify decay intermediates from the 3'-end of albumin mRNA, and as a final proof of principle it was employed to identify in vivo decay intermediates of the c-myc coding region instability determinant corresponding to sites of in vitro cleavage by a polysome-associated endonuclease.