Specific endonucleolytic cleavage sites for decay of Escherichia coli mRNA

The polycistronic lac mRNA of Escherichia coli contains three messages. The rate of degradation of the second (lacY) message was observed to be equal to that of the third (lacA), and each decayed twice as fast as did the first (lacZ). Specific 5'- and 3'-ended lacY mRNA molecules could be recovered from cells; most likely, they are generated from endonucleolytic cleavages that are a part of the degradative process. They were observed by S1 nuclease mapping, and the exact 5'- and 3'-end oligonucleotides of many of them were identified by direct sequencing. Almost all of the molecules started with a 5' adenosine that would be preceded by a pyrimidine. The specificity was further restricted by neighboring nucleotides, and analysis of the data suggested that 5'-U-U decreases-A-U- is especially vulnerable. Also, computer analyses predicted the most stable secondary structures of selected segments of the mRNA and suggested that cleavages may only occur in regions of single strandedness. A model of mRNA degradation is proposed based on these observations and earlier ones. There is no unique target on a message for the initial inactivating attack: any region free of ribosomes is vulnerable, but for statistical reasons the initial attack of most molecules is near the ribosome-loading site. With no further ribosome loading, the newly unprotected 5' ends are "chopped off" at one of the next preferred target sites almost as fast as the last ribosomes moves down the mRNA.     

Specific endonucleolytic cleavages of mouse albumin mRNA and their modulation during liver development.

We have detected specific endonucleolytic cleavages of mouse albumin mRNA by S1 nuclease protection analysis of total RNA from fetal mouse liver using a cDNA probe spanning the middle, coding region of albumin mRNA. With the use of probe labeled at its 5' end, three prominent cleavages were detected which were confirmed and their endonucleolytic nature was established by further analysis using 3' end-labeled probe. The latter probe also revealed one more cleavage which was not detected with the 5' end-labeled probe. These cleavages mapped to positions on the mRNA which included a unique sequence motif CCAN1-3CUGN0-1UGAU. Degradation intermediates corresponding to these cleavages were consistently observed, specifically in fetal liver but not in normal or regenerating adult liver and appeared to have originated in vivo. Their levels decreased progressively from 18th day of gestation and became undetectable by 20 days after birth. No detectable changes in the levels of any of the prominent degradation products of alpha-fetoprotein (a homologue of albumin) mRNA could be observed during this period of development. Since accumulation of degradation intermediates is known to correlate with higher rate of mRNA turnover, our observations raise the possibility that the stability of albumin mRNA may be lower in fetal than in adult mouse liver.     

Degradation of Escherichia coli uncB mRNA by multiple endonucleolytic cleavages.

The mechanism of segmental decay of the uncB sequence near the 5' end of the 7-kb Escherichia coli unc operon mRNA was investigated. Northern (RNA) blots of mRNA expressed from a plasmid carrying the uncBE portion of the operon revealed that the uncB message was rapidly degraded by multiple internal cleavages which resulted in the formation of at least five discrete species having a common 3' end. Turnover studies indicated that processing rapidly converted all species to the smallest. Identification of the 5' ends by primer extension analysis revealed that the cleavages were made either in the uncB coding region or in the intercistronic region between uncB and uncE, the latter being the most 3' cleavage. An rne mutant strain contained much higher levels of the uncBE message, implying that RNase E, the product of the rne gene, is essential for the normal degradation of uncB, and a number of the 5' ends were not detected in the rne mutant. The cleavage sites in chromosomally encoded unc mRNA were also identified by primer extension. These studies reveal that the segmental decay of the uncB region of unc mRNA occurs rapidly through a series of endonucleolytic cleavages. The rapid decay of uncB is expected to play a role in limiting expression of this gene relative to that of the other genes of the operon.     

Site-specific endonucleolytic cleavages and the regulation of stability of E. coli ompA mRNA

The stability of ompA mRNA is growth-rate dependent. We show that the 5' noncoding region of this mRNA provides a target for site-specific endonucleases. The rate of degradation of ompA mRNA parallels the rate of these endonucleolytic cleavages, implying that endonucleolytic rather than exonucleolytic attack is the initial step in ompA mRNA degradation. Thus the 5' noncoding region appears to be a determinant of mRNA stability, and endonucleolytic cleavages in the 5' noncoding region may well regulate expression of the ompA gene.     

Nuclease activity associated with mammalian mRNA in its native state: possible basis for selectivity in mRNA decay.

Polysome and messenger ribonucleoprotein (mRNP) preparations from various mammalian cells contain tightly bound nuclease activity that causes degradation of the mRNA in the preparations. This activity was found to cosediment with all polysome size classes as well as with free mRNPs and to remain associated with the mRNPs released from polysomes by treatment with EDTA. No association with ribosomal subunits was evident. The rates of mRNA degradation were not affected by serial dilution, an indication that enzyme and substrate are tightly associated. beta-Globin mRNA in purified reticulocyte polysomes was cleaved at AU sequences in the 3'-terminal region. Cleavages at the same sites occurred when deproteinized reticulocyte RNA was incubated with mouse sarcoma 180 (S-180) polysomes. The S-180 preparations caused additional cleavages, primarily at UG sequences. A P40 mRNA in S-180 polysomes was cleaved primarily in the 3' noncoding region, but the cleavages in a P21 mRNA were seen in the 5' noncoding region only. Actin mRNA was cleaved in an internal region, yielding large relatively stable 3'- and 5'-terminal fragments. These data suggest the occurrence of highly specific interactions between one or more mRNA-bound nucleases and individual mRNA species.     

Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases

Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.     

Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo

The sorting of the Ash1 protein to the daughter nucleus of Saccharomyces cerevisiae in late anaphase of the budding cycle correlates with the localization of ASH1 mRNA at the bud tip [1] [2]. Although the 3' untranslated region (3' UTR) of ASH1 is sufficient to localize a reporter mRNA, it is not necessary, a result which indicates that other sequences are involved [1]. We report the identification of three additional cis-acting elements in the coding region. Each element alone, when fused to a lacZ reporter gene, was sufficient for the localization of the lacZ mRNA reporter to the bud. A fine-structure analysis of the 3' UTR element showed that its function in mRNA localization did not depend on a specific sequence but on the secondary and tertiary structure of a minimal 118 nucleotide stem-loop. Mutations in the stem-loop that affect the localization of the lacZ mRNA reporter also affected the formation of the localization particles, in living cells, composed of a green fluorescent protein (GFP) complexed with lacZ-ASH1-3' UTR mRNA [3]. A specific stem-loop in the 3' UTR of the ASH1 mRNA is therefore required for both localization and particle formation, suggesting that complex formation is part of the localization mechanism. An analysis on one of the coding-region elements revealed a comparable stem-loop structure with similar functional requirements.     

The sequence of the stem and flanking sequences at the 3' end of histone mRNA are critical determinants for the binding of the stem-loop binding protein.

Complexes of different electrophoretic mobility containing the stem-loop binding protein, a 45 kDa protein, bound to the stem-loop at the 3' end of histone mRNA, are present in both nuclear and cytoplasmic extracts from mammalian cells. We have determined the effect of changes in the loop, in the stem and in the flanking sequences on the affinity of the SLBP for the 3' end of histone mRNA. The sequence of the stem is particularly critical for SLBP binding. Specific sequences both 5' and 3' of the stem-loop are also required for high-affinity binding. Expanding the four base loop by one or two uridines reduced but did not abolish SLBP binding. RNA footprinting experiments show that the flanking sequences on both sides of the stem-loop are critical for efficient binding, but that cleavages in the loop do not abolish binding. Thus all three regions of the RNA sequence contribute to SLBP binding, suggesting that the 26 nt at the 3' end of histone mRNA forms a defined tertiary structure recognized by the SLBP.