Truncation of exon 1 from the c-myc gene results in prolonged c-myc mRNa stability.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32P04 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.We have previously demonstrated that the mRNA population in vegetatively growing amoebae of the cellular slime mold Dictyostelium discoideum decays with complex kinetics, consisting of at least two major components: a rapidly decaying component with a half-life of approximately 50 min, and a long-lived component with a half-life of approximately 10 h (14, 58). Similar complex kinetics have been observed for the decay of poly(A)+ RNA in a variety of other eucaryotic cells (5,25,39,57,77,78,81). It is likely that these complex decay kinetics reflect the sum of the decay rates of individual mRNAs (e.g., in D. discoideum, the most stable mRNAs have half-lives of approximately 10 h and the least stable mRNAs have half-lives which are approximately 10-fold shorter). It has been s...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

Shapiro¹,

Herrick²,

Manrow³

et al. 1988

“…Chicken c-mvc RNA can also potentially form a stem structure by annealing portions of exons 1 and 2 with a minimum free energy of AGO (250) of -45 kcal/mol (44) which is not as low as in the human RNA stem (-90 kcal/mol). More likely, removal of exon 1 affects RNA stability (15,34). Thus, the removal of exon 1 in most retrovirus-induced bursal lymphomas (11,37), as well as in the majority of c-myc-immunoglobulin locus translocations found in plasmacytomas (3,20,41,43) and some Burkitt's lymphomas (3,5), may enhance translation of c-myc (11,37).…”

Section: (40)mentioning

confidence: 99%

Features of the chicken c-myc gene that influence the structure of c-myc RNA in normal cells and bursal lymphomas.

Nottenburg¹,

Varmus²

1986

The chicken c-myc gene is the target for proviral insertion mutations in bursal lymphomas and has been transduced to generate several viral oncogenes, but the boundaries of its exons have not been securely established. To define the landmarks of the chicken c-myc gene necessary to produce its mRNA, we used an RNase protection assay and a cDNA clone to analyze the c-myc mRNAs from normal chicken embryos and from two bursal lymphomas: LL6, which contains an avian leukosis virus provirus downstream of the c-myc coding region, and LL7, which contains an avian leukosis virus provirus upstream of the c-myc coding region. Two initiation sites for normal c-myc mRNA are less than 7 bases apart, downstream of a GC-rich region lacking canonical TATA and CAAT sequences. The first exon has two open reading frames for the entire length but no initiator methionine codons. The splice donor and acceptor sites at the boundary of the first intron were assigned by comparing a sequence of an LL6 c-myc cDNA clone with a genomic DNA sequence and confirmed by RNase protection of labeled RNA probes by normal and LL6-derived mRNAs. Two potential polyadenylation signals are located approximately 250 and 400 bases downstream of the c-myc coding region in the third exon, but only the more distal signal is utilized in both normal cells and the LL7 tumor. The proviral integration in the LL6 tumor occurred upstream of the authentic c-myc polyadenylation signal accounting for polyadenylation of this transcript in the proviral long terminal repeat.

“…Due to translocation, the first untranslated exon of the normal c-myc mRNA is often replaced with other shorter 5' untranslated sequences (7). It has been demonstrated by several groups that this truncated c-myc mRNA has a longer half-life than normal full-length c-myc mRNA (13,32,34), suggesting that part of the signal for c-myc mRNA degradation may reside in exon 1. The increased stability of truncated c-myc transcripts contributes to the increased levels of c-myc transcripts which are probably casual in the malignant transformation of these B-cell tumors.…”

mentioning

confidence: 99%

Differential stability of c-myc mRNAS in a cell-free system.

Pei

Calame

1988

We have developed a simple cell-free system for studying the stability of different mRNAs in vitro. We demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin Ca heavy chain or glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.