Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.     

Transcript-specific decapping and regulated stability by the human Dcp2 decapping protein

mRNA decapping is a critical step in the control of mRNA stability and gene expression and is carried out by the Dcp2 decapping enzyme. Dcp2 is an RNA binding protein that must bind RNA in order to recognize the cap for hydrolysis. We demonstrate that human Dcp2 (hDcp2) preferentially binds to a subset of mRNAs and identify sequences at the 5' terminus of the mRNA encoding Rrp41, a core subunit component of the RNA exosome, as a specific hDcp2 substrate. A 60-nucleotide element at the 5' end of Rrp41 mRNA was identified and shown to confer more efficient decapping on a heterologous RNA both in vitro and upon transfection into cells. Moreover, reduction of hDcp2 protein levels in cells resulted in a selective stabilization of the Rrp41 mRNA, confirming it as a downstream target of hDcp2 regulation. These findings demonstrate that hDcp2 can specifically bind to and regulate the stability of a subset of mRNAs, and its intriguing regulation of the 3'-to-5' exonuclease exosome subunit suggests a potential interplay between 5'-end mRNA decapping and 3'-end mRNA decay.     

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3' ends of mature mRNAs. This is accompanied by trans-splicing with SL2 approximately 100 nucleotides downstream of the 3' end formation sites to create the 5' ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located approximately 70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3' end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3' end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5' or 3' direction alters the location of the 5' end of the Ur-RNA. We propose that a 5' to 3' exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies.     

The cleavage/polyadenylation activity triggered by a U-rich motif sequence is differently required depending on the poly(A) site location at either the first or last 3'-terminal exon of the 2'-5' oligo(A) synthetase gene

Production of the two mRNAs encoding distinct forms of 2'-5'-oligoadenylate synthetase depends on processing that involves the recognition of alternative poly(A) sites and an internal 5'-splice site located within the first 3'-terminal exon. The resulting 1.6- and 1.8-kb mRNAs are expressed in fibroblast cell lines, whereas lymphoblastoid B cells, such as Daudi, produce only the 1.8-kb mRNA. In the present study, we have shown that the 3'-end processing at the last 3'-terminal exon occurs independently of the core poly(A) site sequence or the presence of regulatory elements. In contrast, in Daudi cells, the recognition of the poly(A) site at the first 3'-terminal exon is impaired because of an unfavorable sequence context. The 3'-end processing at this particular location requires a strong stabilization of the cleavage/polyadenylation factors, which can be achieved by the insertion of a 25-nucleotide long U-rich motif identified upstream of the last poly(A) site. Consequently, we speculate that in cells expressing the 1.6-kb mRNA, such as fibroblasts, direct or indirect participation of a specific mechanism or cell type-specific factors are required for an efficient polyadenylation at the first 3'-terminal exon.     

In vivo studies on putative Shine-Dalgarno sequences of the halophilic archaeon Halobacterium salinarum

The involvement of Shine-Dalgarno sequences in the translation of mRNA in halophilic archaea was investigated for two gvp genes involved in gas vesicle formation in Halobacterium salinarum PHH1. With the exception of gvpA and gvpO, all reading frames of the p-gvpDEFGHIJKLM and p-gvpACNO mRNAs contained upstream of the AUG start codon a putative Shine-Dalgarno (SD) sequence that is complementary to the 3'-end of the small ribosomal subunit RNA. The importance of the SD sequences of gvpG and gvpH was investigated in Haloferax volcanii transformants, and an alteration of the SD sequence resulted in a reduction of the amount of the GvpG or GvpH protein. For a more quantitative analysis the region upstream of gvpH was fused to the bgaH reading frame encoding an enzyme with beta-galactosidase activity as reporter. Scanning mutagenesis within the mRNA leader demonstrated that mutations adjacent to the putative SD sequence GGAGGUCA did not influence the efficiency of translation, whereas constructs harbouring an altered SD sequence yielded only 5-50% of the beta-galactosidase activities obtained with the wild-type SD element. A complete mutation of the SD sequence still yielded 20% of the wild-type activity. Alterations in the spacing of the SD sequence and the translation initiation codon of gvpH indicated that a distance of 4 or 10 nucleotides yielded a similar beta-galactosidase activity as found with the 7 nt spacing of the SD element in wild type, whereas a distance of 1 nt resulted in the loss of translation. A complete deletion of the 5'-UTR resulting in a leaderless mRNA yielded an enhanced beta-galactosidase activity in transformants implying that the initiation of translation involved a mechanism other than a specific mRNA-rRNA interaction.     

Growth factor-mediated stabilization of amyloid precursor protein mRNA is mediated by a conserved 29-nucleotide sequence in the 3'-untranslated region

Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5'-untranslated region (UTR) and coding region of beta-globin and the entire 3'-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3'-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t 1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t 1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t 1/2 = 40 min). We therefore conclude that the 29-nucleotide 3'-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.     

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation.

We investigated the cis-acting sequences involved in termination of vesicular stomatis virus mRNA synthesis by using bicistronic genomic analogs. All of the cis-acting signals necessary for termination reside within the first 13 nucleotides of the 23-nucleotide conserved gene junction. This 13-nucleotide termination sequence at the end of the upstream gene comprises the tetranucleotide AUAC, the tract containing seven uridines (U7 tract), and the intergenic dinucleotide (GA), but it does not include the downstream gene start sequence. Data presented here show that upstream mRNA termination is independent of downstream mRNA initiation. Alteration of any nucleotide in the 13-nucleotide sequence decreased the termination activity of the gene junction and resulted in increased synthesis of a bicistronic readthrough RNA. This finding indicated that the wild-type gene junction has evolved to achieve the maximum termination efficiency. The most critical position of the AUAC sequence was the C, which could not be altered without complete loss of mRNA termination. Reducing the length of the wild-type U7 tract to zero, five, or six U residues also totally abolished mRNA termination, resulting in exclusive synthesis of the bicistronic readthrough mRNA. Shortening the wild-type U7 tract to either five or six U residues abolished VSV polymerase slippage during readthrough RNA synthesis. Since neither the U5 nor U6 template was able to direct mRNA termination, these data imply that polymerase slippage is a prerequisite for termination. Evidence is also presented to show that in addition to causing polymerase slippage, the U7 tract itself or its poly(A) product constitutes an essential signal for mRNA termination.     

Processed pseudogenes for rat cytochrome c are preferentially derived from one of three alternate mRNAs.

Three cytochrome c mRNAs (1,400, 1,100 and 700 nucleotides) are colinear with RC4, a gene that has introns and correctly encodes cytochrome c. A comparison of RC4 to six nonallelic clones isolated from the rat cytochrome c multigene family demonstrates that all three mRNAs are represented in the genome as processed pseudogenes. Four of the six pseudogenes are derived from the 1,100-nucleotide mRNA, and genomic hybridizations further establish that nearly all of the 30 or so gene family members are also genomic copies of this mRNA despite the equimolar ratio of the three messages in rat tissues. Thus, the surprising multiplicity of cytochrome c sequences in the rat genome is mainly accounted for by the selective use of the 1,100-nucleotide mRNA for the formation of processed pseudogenes. In contrast to 700- and 1,400-nucleotide species which are polyadenylated downstream from AAGUAAA and AAUUAAA, respectively, the 1,100-nucleotide mRNA uses the ubiquitous AAUAAA and also displays a unique stem and loop structure (delta G = -59.4 kJ) centered 37 base pairs upstream from this sequence.     

Determinants of Drosophila fushi tarazu mRNA instability.

The fushi tarazu gene is essential for the establishment of the Drosophila embryonic body plan. When first expressed in early embryogenesis, fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, fushi tarazu mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of fushi tarazu mRNA is probably crucial for attaining this localized pattern of expression. mRNA stability in transgenic embryos was measured by a new method that does not use drugs or external interference. Experiments using hybrid genes that fuse fushi tarazu sequences to those of the stable ribosomal protein A1 mRNA provide evidence for at least two destabilizing elements in the fushi tarazu mRNA, one located within the 5' one-third of the mRNA and the other near the 3' end (termed FIE3 for ftz instability element 3'). The FIE3 lies within a 201-nucleotide sequence just upstream of the polyadenylation signal and can act autonomously to destabilize a heterologous mRNA. Further deletion constructs identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other previously identified destabilization sequences suggests that FIE3 contains a novel RNA destabilization element.     

Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae.

The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.     

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.