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.     

The mammalian exosome mediates the efficient degradation of mRNAs that contain AU-rich elements.

HeLa cytoplasmic extracts contain both 3'-5' and 5'-3' exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modified RNAs. These data indicate that 3'-5' exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immunodepletion using antibodies specific for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for efficient 3'-5' exonucleolytic decay. Furthermore, 3'-5' exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact specifically with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the efficiency of ARE-containing mRNA turnover.     

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.     

The Cth2 ARE-binding protein recruits the Dhh1 helicase to promote the decay of succinate dehydrogenase SDH4 mRNA in response to iron deficiency

Iron is an essential nutrient that participates as a redox co-factor in a broad range of cellular processes. In response to iron deficiency, the budding yeast Saccharomyces cerevisiae induces the expression of the Cth1 and Cth2 mRNA-binding proteins to promote a genome-wide remodeling of cellular metabolism that contributes to the optimal utilization of iron. Cth1 and Cth2 proteins bind to specific AU-rich elements within the 3'-untranslated region of many mRNAs encoding proteins involved in iron-dependent pathways, thereby promoting their degradation. Here, we show that the DEAD box Dhh1 helicase plays a crucial role in the mechanism of Cth2-mediated mRNA turnover. Yeast two-hybrid experiments indicate that Cth2 protein interacts in vivo with the carboxyl-terminal domain of Dhh1. We demonstrate that the degradation of succinate dehydrogenase SDH4 mRNA, a known target of Cth2 on iron-deficient conditions, depends on Dhh1. In addition, we localize the Cth2 protein to cytoplasmic processing bodies in strains defective in the 5' to 3' mRNA decay pathway. Finally, the degradation of trapped SDH4 mRNA intermediates by Cth2 supports the 5' to 3' directionality of mRNA turnover. Taken together, these results suggest that Cth2 protein recruits the Dhh1 helicase to ARE-containing mRNAs to promote mRNA decay.     

Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.     

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 digested in 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.     

The complete nucleotide sequence of human liver cytochrome b5 mRNA

We have isolated and sequenced a cDNA clone corresponding to human liver cytochrome b5 mRNA. The 760 base pair (bp) sequence contains the complete coding and 3' non-translated regions plus 52 bp of 5' non-translated sequence. The derived amino acid sequence showed that the previous assignment of several amino acids was in error. In addition, the sequence of the previously unknown COOH hydrophobic region has been obtained.     

CD28-Mediated regulation of mRNA stability requires sequences within the coding region of the IL-2 mRNA.

Using sequence-tagged genomic reporter constructs, we investigated the contribution of IL-2 sequences to CD28-mediated regulation of mRNA stability. We find that CD28 signaling acts transiently to stabilize the IL-2 mRNA following T cell activation. Such stabilization requires sequences within both exon 2 and the coding region of exon 4. Unexpectedly, CD28 signaling at later times enhances the decay of the IL-2 mRNA. This CD28-dependent decay of IL-2 mRNA requires sequences localized between exon 3 and the stop codon. Our findings demonstrate that the coding region of the IL-2 mRNA contains previously undefined CD28-responsive sequence elements that are critical for the regulation of mRNA stability.     

The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1

An important control step in the regulation of cytoplasmic mRNA turnover is the removal of the m(7)G cap structure at the 5' end of the message. Here, we describe the functional characterization of Dhh1, a highly conserved member of the family of DEAD box-containing proteins, as a regulator of mRNA decapping in Saccharomyces cerevisiae. Dhh1 is a cytoplasmic protein and is shown to be in a complex with the mRNA degradation factor Pat1/Mtr1 and with the 5'-3' exoribonuclease Xrn1. Dhh1 specifically affects mRNA turnover in the deadenylation-dependent decay pathway, but does not act on the degradation of nonsense-containing mRNAs. Cells that lack dhh1 accumulate degradation intermediates that have lost their poly(A) tail but contain an intact 5' cap structure, suggesting that Dhh1 is required for efficient decapping in vivo. Furthermore, recombinant Dhh1 is able to stimulate the activity of the purified decapping enzyme Dcp1 in an in vitro decapping assay. We propose that the DEAD box protein Dhh1 regulates the access of the decapping enzyme to the m(7)G cap by modulating the structure at the 5' end of mRNAs.