Mark N. Hanson

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.

Apparent defects in processive DNA synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse transcriptase derive solely from a dNTP utilization defect.

The 2′,3′-dideoxy-3′-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3′-end-directed and RNA 5′-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.

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 digestedin 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.

Mechanism Analysis Indicates that Recombination Events in HIV-1 Initiate and Complete Over Short Distances, Explaining Why Recombination Frequencies Are Similar in Different Sections of the Genome

Strand transfer drives recombination between the co-packaged genomes of HIV-1, a process that allows rapid viral evolution. The proposed invasion-mediated mechanism of strand transfer during HIV-1 reverse transcription has three steps: (1) invasion of the initial or donor primer template by the second or acceptor template; (2) propagation of the primer-acceptor hybrid; and (3) primer terminus transfer. Invasion occurs at a site at which the reverse transcriptase ribonuclease H (RNase H) has created a nick or short gap in the donor template. We used biochemical reconstitution to determine the distance over which a single invasion site can promote transfer. The DNA-primed RNA donor template used had a single-stranded pre-created invasion site (PCIS). Results showed that the PCIS could influence transfer by 20 or more nucleotides in the direction of synthesis. This influence was augmented by viral nucleocapsid protein and additional reverse transcriptase-RNase H cleavage. Strand-exchange assays were performed specifically to assess the distance over which a hybrid interaction initiated at the PCIS could propagate to achieve transfer. Propagation by simple branch migration of strands was limited to 24-32 nt. Additional RNase H cuts in the donor RNA allowed propagation to a maximum distance of 32-64 nt. Overall, results indicate that a specific invasion site has a limited range of influence on strand transfer. Evidently, a series of invasion sites cannot collaborate over a long distance to promote transfer. This result explains why the frequency of recombination events does not increase with increasing distance from the start of synthesis, a characteristic that supports effective mixing of viral mutations.

Proximity and Branch Migration Mechanisms in HIV-1 Minus Strand Strong Stop DNA Transfer

Human immunodeficiency virus type 1 minus strand transfer was measured using a genomic donor-acceptor template system in vitro. Donor RNA D199, having the minimum region required for minus strong stop DNA synthesis, was previously shown to transfer with 35% efficiency to an acceptor RNA representing the 3′ repeat region. Donor D520, having an additional 321-nucleotide segment extending into gag, transferred at 75% efficiency. In this study each transfer step was analyzed to account for the difference. Measurement of terminal transfer indicated that the 3′ terminus of the cDNA generated using D520 is more accessible for transfer than that of D199. Nevertheless, acceptor competition experiments demonstrated that D520 has a greater preference for invasion-driven versus terminal transfer than D199. Competition mapping showed that the base of the transactivation response element is the primary invasion site for D520, important for efficient acceptor invasion. Acceptors complementary to the invasion and terminal transfer sites, but not the region between, allowed assessment of the significance of hybrid propagation by branch migration. These bipartite acceptors showed that with D520, invasion raises the local concentration of the acceptor for efficient terminal transfer by a proximity effect. However, with D199, invasion is relatively inefficient, and the cDNA 3′ terminus is not very accessible. For most transfers that occurred, the acceptor accessed the cDNA 3′ end by branch migration. Results suggest that both proximity and branch migration mechanisms contribute to transfers, with the proportion determined by donor-cDNA structure. D520 transfers better because it has greater accessibility for both invasion and terminus transfer.

Polysomal Ribonuclease 1

Publisher Summary This chapter describes methods and approaches to purify and characterize polysomal ribonuclease 1 (PMR-1). It is the first mRNA endoribonuclease to be purified and cloned. Because PMR-1 was found in association with mRNA-containing complexes, the first steps in its analysis required facile approaches for the isolation of messenger ribonucleoprotein (mRNP) and polysome complexes. Furthermore, PMR-1 selectively associates with membrane-bound polysomes. Two approaches are employed to test whether PMR-1 (or another endonuclease) is specific for single-stranded RNA, or whether it can cleave double-stranded RNA. PMR-1 associates with substrate mRNA in vivo in an RNP complex. Two methods were used to recover these complexes; selection of poly(A)-containing complexes with oligo(dT)-cellulose, and immunoprecipitation of PMR-1-containing complexes with a polyclonal antibody to PMR-1. There is convincing evidence of the appearance of PMR-1 cleavage sites in albumin mRNA coincident with the estrogen-induced initiation of albumin mRNA decay. Thus, the protocols developed for this mRNA endonuclease should be generally applicable to the characterization of other mRNA endonucleases as they are identified.