Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3'' terminus.

The first detectable step in the degradation of human H4 histone mRNA occurs at the 3' terminus in a cell-free mRNA decay system (J. Ross and G. Kobs, J. Mol. Biol. 188:579-593, 1986). Most or all of the remainder of the mRNA is then degraded in a 3'-to-5' direction. The experiments described here were designed to determine whether a similar degradation pathway is followed in whole cells. Two sets of short-lived histone mRNA decay products were detected in logarithmically growing erythroleukemia (K562) cells. These products, designated the -5 and -12 RNAs, were generated by the loss of approximately 4 to 6 and 11 to 13 nucleotides, respectively, from the 3' terminus of histone mRNA. The same decay products were observed after a brief incubation in vitro. They were in low abundance or absent from cells that were not degrading histone mRNA. In contrast, they were readily detectable in cells that degraded the mRNA at an accelerated rate, i.e., in cells cultured with a DNA synthesis inhibitor, either cytosine arabinoside or hydroxyurea. During the initial stages of the decay process, as the 3' terminus of the mRNA was being degraded, the 5'-terminal region remained intact. These results indicate that the first detectable step in human H4 histone mRNA decay occurs at the 3' terminus and that degradation proceeds 3' to 5', both in cells and in cell-free reactions.     

Protein synthesis inhibition stabilizes urokinase-type plasminogen activator mRNA. Studies in vivo and in cell-free decay reactions

Inhibition of protein synthesis stabilizes a number of mRNAs, but little is known about the mechanism. To understand the relationship between protein synthesis and mRNA stability, we studied the degradation of calcitonin-induced urokinase-type plasminogen activator (uPA) mRNA in LLC-PK cells. uPA mRNA became highly stable by pretreatment with either cycloheximide or pactamycin, and the stabilizing effect of cycloheximide treatment was time dependent with the full effect exerted by 60 min. Stabilization was also observed with histone H4 mRNA but only partially with c-myc mRNA. To further analyze, we developed a cell-free decay reaction system based on post-mitochondrial supernatant (PMS). In this system, uPA mRNA was completely stable when fractions were obtained from cells pretreated with cycloheximide, but very unstable in control fractions, paralleling uPA mRNA stability in intact cells. However, in contrast to uPA mRNA and the in vivo observation, histone H4 mRNA was unstable whether or not the cells were pretreated with cycloheximide. These results suggest that inhibition of protein synthesis stabilizes mRNAs in at least two different ways in LLC-PK1 cells. When PMS from cycloheximide/calcitonin-treated cells was mixed with PMS from untreated cells, uPA mRNA was not destabilized. This suggests that a putative labile factor responsible for uPA mRNA degradation is not a soluble protein.     

Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.     

5' to 3' exoribonucleolytic activity is a normal component of chloroplast mRNA decay pathways

Molecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5' and 3' untranslated regions. While it is well-known that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G)18 sequence, which impedes both 5'-->3' and 3'-->5' exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions -20, +25, +165 or +25/+165 relative to the mature petD 5' end, mRNAs accumulate with 5' ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5' end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5'-->3' exoribonucleolytic activity. In the case of the -20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5' end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5' ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) -20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.     

Faithful degradation of soybean rbcS mRNA in vitro.

The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.     

Regulated alpha-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping

The alpha-globin mRNA contains a C-rich stability element (CRE) in its 3' untranslated region (3' UTR) which is critical for the stability of this long-lived mRNA. A protein complex, termed the alpha-complex, forms on the CRE and has been shown to contribute to stabilization of the mRNA by at least two mechanisms, first by interacting with the poly(A)-binding protein (PABP) to prevent deadenylation, and second by protecting the mRNA from attack by an erythroid endoribonuclease. In this report, we demonstrate that the alpha-globin 3' UTR can confer stability on a heterologous mRNA in cells, and this stability is dependent on the alpha-complex. Moreover, the stability was exclusively detected with cytoplasmic mRNA, suggesting that the regulation of alpha-globin mRNA stability is a cytoplasmic event. An additional mechanism by which the alpha-complex can confer stability on an RNA in vitro was also identified and shown to involve inhibition of 3' to 5' exonucleolytic degradation. Furthermore, using an in vitro mRNA decay system, we were able to follow the demise of the alpha-globin RNA and demonstrate that the decay was initiated by deadenylation followed by 3'-to-5' decay carried out by the exosome and ultimately hydrolysis of the residual cap structure.     

Poly(A) shortening and degradation of the 3'' A+U-rich sequences of human c-myc mRNA in a cell-free system.

The early steps in the degradation of human c-myc mRNA were investigated, using a previously described cell-free mRNA decay system. The first detectable step was poly(A) shortening, which generated a pool of oligoadenylated mRNA molecules. In contrast, the poly(A) of a stable mRNA, gamma globin, was not excised, even after prolonged incubation. The second step, degradation of oligoadenylated c-myc mRNA, generated decay products whose 3' termini were located within the A+U-rich portion of the 3' untranslated region. These products disappeared soon after they were formed, consistent with rapid degradation of the 3' region. In contrast, the 5' region, corresponding approximately to c-myc exon 1, was stable in vitro. The data indicate a sequential degradation pathway in which 3' region cleavages occur only after most or all of the poly(A) is removed. To account for rapid deadenylation, we suggest that the c-myc poly(A)-poly(A)-binding protein complex is readily dissociated, generating a protein-depleted poly(A) tract that is no longer resistant to nucleases.     

Evidence for a 3'-5' decay pathway for c-myc mRNA in mammalian cells.

Many mRNAs in mammalian cells decay via a sequential pathway involving rapid conversion of polyadenylated molecules to a poly(A)-deficient state followed by rapid degradation of the poly(A)-deficient molecules. However, the rapidity of this latter step(s) has precluded further analyses of the decay pathways involved. Decay intermediates derived from degradation of poly(A)-deficient molecules could offer clues regarding decay pathways, but these intermediates have not been readily detected. Cell-free mRNA decay systems have proven useful in analyses of decay pathways because decay intermediates are rather stable in vitro. Cell-free systems indicate that many mRNAs decay by a sequential 3'-5' pathway because 3'-terminal decay intermediates form following deadenylation. However, if 3'-terminal, in vitro decay intermediates reflect a biologically significant aspect of mRNA turnover, then similar intermediates should be present in cells. Here, I have compared the in vivo and in vitro decay of mRNA encoded by the c-myc proto-oncogene. Its decay both in vivo and in vitro occurs by rapid removal of the poly(A) tract and generation of a 3'-terminal decay intermediate. These data strongly suggest that a 3'-5' pathway contributes to turnover of c-myc mRNA in cells. It is likely that 3'-5' decay represents a major turnover pathway in mammalian cells.     

Functional link between the mammalian exosome and mRNA decapping.

Mechanistic understanding of mammalian mRNA turnover remains incomplete. We demonstrate that the 3' to 5' exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract. An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap. The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA. These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.     

Importance of a 5′ Stem-Loop for Longevity of papA mRNA in Escherichia coli

High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal.     

Terminal sequences do not contain the rate-limiting decay determinants of E. coli cat mRNA.

The mechanism of E. coli chloramphenicol acetyltransferase (cat) mRNA decay was investigated. Alteration of the 5' untranslated terminus does not appear to have an effect on the turnover rate of the mRNA. Similarly, changes at the 3' terminus of the message, including the addition of a stable stem and loop structure, do not affect the half-life of the message. The data suggest that 5' and 3' terminal untranslated sequences do not contain the rate-limiting determinants for cat message decay. Decay rates for various segments of the cat mRNA were measured and indicate that all regions of the message have similar stabilities. The current model of cat mRNA degradation involves a rate-limiting endonucleolytic decay event that occurs internal to the message followed by degradation of the cleavage products.     

Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Degradation of the soybean ribulose-1,5-bisphosphate carboxylase small-subunit mRNA, SRS4, initiates with endonucleolytic cleavage.

The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.     

Multiple mRNA decapping enzymes in mammalian cells

Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5' end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover.