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.     

Degradation products of the mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean and transgenic petunia.

The degradation of a soybean ribulose-1,5-bisphosphate carboxylase small subunit RNA, SRS4, was investigated in soybean seedlings and in petunia plants transformed with an SRS4 gene construct. Polyacrylamide RNA gel blot, primer extension, and S1 nuclease analyses were used to identify and map fragments of the SRS4 mRNA generated in vivo. We showed that SRS4 mRNA is degraded to a characteristic set of fragments in soybean and transgenic petunia and that degradation is not dependent on position of insertion of the gene construct within the genome, on the expression level of the SRS4 mRNA, or on the rbcS promoter. Degradation products lacked poly(A) tails and fractionated with poly(A)-depleted RNA on oligo(dT)-sepharose columns. These products pelleted with polysomes and were released from polysomes prepared with EDTA. Sequences at the 5' end of the SRS4 mRNA were more stable than those at the 3' end of the mRNA. Three models for SRS4 mRNA degradation involving endonucleolytic and exonucleolytic degradation were presented to explain the origin of the 5' proximal fragments.     

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.     

H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'

The relative decay of four human messenger RNAs, gamma globin, delta globin, c-myc and H4 histone, were compared in a cell-free system. Under appropriate conditions, they are degraded in vitro in approximately the same relative order as in vivo: histone faster than c-myc and delta globin faster than gamma globin. Degradation of polysome-associated H4 histone mRNA and of deproteinized histone mRNA begins at or near the 3' terminus. At least a portion of the mRNA then continues to be degraded in a 3' to 5' direction. Discrete 3'-terminal degradation hold-up points are observed, suggesting that 3' to 5' degradation occurs non-uniformly. Cycloheximide and puromycin inhibit protein synthesis but do not affect the rate or directionality of histone mRNA decay in vitro. We conclude that the rate-limiting step in H4 histone mRNA decay occurs at or near the 3' terminus and that at least a portion of the mRNA molecule is subsequently degraded 3' to 5', probably via a processive exonuclease.     

Further characterization of mRNA''s of mouse hepatitis virus: presence of common 5''-end nucleotides

The mouse hepatitis virus strain A59 codes for seven RNA species in the infected cells. These virus-specific RNAs were found to be polysome associated and therefore likely to represent mRNA's. All of them have common 3'-end sequences (Lai et al., J. Virol. 39:823-834, 1981). Their structure was further studied with respect to their 5'-end sequences. It was found that all of these mRNA's contained cap structures at their 5' ends. Furthermore, the cap-containing oligonucleotides which represent the sequences immediately adjacent to the 5' ends were found to be the same for most, if not all, of the seven virus-specific mRNA's. These sequences are also identical to the 5'-end sequences of the virion RNA genome. The 5'-end sequences were tentatively determined to be 5'-cap-N-UAAG. The presence of the common nucleotides in all of the virus-specific RNAs in mouse hepatitis virus strain A59 suggests several possible mechanisms of synthesis for these RNAs. The significance of these findings is discussed.     

Polyadenylation accelerates degradation of chloroplast mRNA.

The expression of chloroplast genes is regulated by several mechanisms, one of which is the modulation of RNA stability. To understand how this regulatory step is controlled during chloroplast development, we have begun to define the mechanism of plastid mRNA degradation. We show here that the degradation petD mRNA involves endonucleolytic cleavage at specific sites upstream of the 3' stem-loop structure. The endonucleolytic petD cleavage products can be polyadenylated in vitro, and similar polyadenylated RNA products are detectable in vivo. PCR analysis of the psbA and psaA-psaB-rps14 operons revealed other polyadenylated endonucleolytic cleavage products, indicating that poly(A) addition appears to be an integral modification during chloroplast mRNA degradation. Polyadenylation promotes efficient degradation of the cleaved petD RNAs by a 3'-5' exoribonuclease. Furthermore, polyadenylation also plays an important role in the degradation of the petD mRNA 3' end. Although the 3' end stem-loop is usually resistant to nucleases, adenylation renders the secondary structure susceptible to the 3'-5' exoribonuclease. Analysis of 3' ends confirms that polyadenylation occurs in vivo, and reveals that the extent of adenylation increases during the degradation of plastid mRNA in the dark. Based on these results, we propose a novel mechanism for polyadenylation in the regulation of plastid mRNA degradation.     

Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro.

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.