Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay

No-go decay (NGD) is one of several messenger RNA (mRNA) surveillance systems dedicated to the removal of defective mRNAs from the available pool. Two interacting factors, Dom34 and Hbs1, are genetically implicated in NGD in yeast. Using a reconstituted yeast translation system, we show that Dom34:Hbs1 interacts with the ribosome to promote subunit dissociation and peptidyl-tRNA drop-off. Our data further indicate that these recycling activities are shared by the homologous translation termination factor complex eRF1:eRF3, suggesting a common ancestral function. Because Dom34:Hbs1 activity exhibits no dependence on either peptide length or A-site codon identity, we propose that this quality-control system functions broadly to recycle ribosomes throughout the translation cycle whenever stalls occur.     

Translation of the edited mRNA for cytochrome b in trypanosome mitochondria

The type of RNA editing found in the kinetoplast-mitochondria of trypanosomes and related protozoa, involving uridylate insertions and deletions, creates translatable messenger RNAs (mRNAs) out of nonsense pre-edited RNAs by correcting encoded defects that vary from simple frameshifts to large "cryptic" regions. However, any evidence for translation of these mRNAs in the kinetoplast has been missing for decades. We identified a kinetoplast-encoded protein, apocytochrome b, whose mRNA is edited in the 5' region. The determined amino-terminal sequence of the protein coincides with the predicted sequence derived from the edited region, demonstrating that the cognate apocytochrome b mRNA is translated into a functional protein. This finding represents the first direct evidence for a functional translation system in the kinetoplasts.     

Translational blockade imposed by cytokine-derived UA-rich sequences

The messenger RNAs specifying certain proteins involved in the inflammatory response and certain oncoproteins contain a conserved UA-rich sequence in the 3' untranslated region. This sequence, which is composed of several interspersed repeats of the octanucleotide UUAUUUAU, has been shown to destabilize mRNA in some eukaryotes. However, this effect is not seen when mRNAs are transferred to Xenopus oocytes, which made it possible to separate stability from translational regulation. For interferon, granulocyte-macrophage colony-stimulating factor, and c-fos RNAs, the UA-rich sequence was observed to preclude mRNA translation.     

Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex

Nonsense-mediated messenger RNA (mRNA) decay, or NMD, is a critical process of selective degradation of mRNAs that contain premature stop codons. NMD depends on both pre-mRNA splicing and translation, and it requires recognition of the position of stop codons relative to exon-exon junctions. A key factor in NMD is hUpf3, a mostly nuclear protein that shuttles between the nucleus and cytoplasm and interacts specifically with spliced mRNAs. We found that hUpf3 interacts with Y14, a component of post-splicing mRNA-protein (mRNP) complexes, and that hUpf3 is enriched in Y14-containing mRNP complexes. The mRNA export factors Aly/REF and TAP are also associated with nuclear hUpf3, indicating that hUpf3 is in mRNP complexes that are poised for nuclear export. Like Y14 and Aly/REF, hUpf3 binds to spliced mRNAs specifically ( approximately 20 nucleotides) upstream of exon-exon junctions. The splicing-dependent binding of hUpf3 to mRNAs before export, as part of the complex that assembles near exon-exon junctions, allows it to serve as a link between splicing and NMD in the cytoplasm.     

Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction

Iron-responsive elements (IREs) are RNA motifs that have been identified within the 5' untranslated region of ferritin messenger RNA and the 3' untranslated region of transferrin receptor mRNA. A single IRE mediates iron-dependent control of ferritin translation, whereas multiple IREs are found in the region of the transferrin receptor mRNA responsible for iron-dependent control of mRNA stability. A cytosolic protein binds in vitro to the IREs of both mRNAs. The IRE-binding protein (IRE-BP) is shown to require free sulfhydryl groups for its specific interaction with the IRE. Treatment of lysates with reducing agents increases the binding activity, whereas agents that block sulfhydryls inhibit binding. Iron starvation, leading to decreased ferritin translation, results in increased binding activity, which is explained by an increase in the fraction of the IRE-BP that is in a fully reduced state.     

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish

MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.     

miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay

microRNAs (miRNAs) regulate gene expression through translational repression and/or messenger RNA (mRNA) deadenylation and decay. Because translation, deadenylation, and decay are closely linked processes, it is important to establish their ordering and thus to define the molecular mechanism of silencing. We have investigated the kinetics of these events in miRNA-mediated gene silencing by using a Drosophila S2 cell-based controllable expression system and show that mRNAs with both natural and engineered 3' untranslated regions with miRNA target sites are first subject to translational inhibition, followed by effects on deadenylation and decay. We next used a natural translational elongation stall to show that miRNA-mediated silencing inhibits translation at an early step, potentially translation initiation.     

Quality control mechanisms during translation

Translation uses the genetic information in messenger RNA (mRNA) to synthesize proteins. Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. The cycle is then repeated to produce a full-length protein. Proofreading and editing processes are used throughout protein synthesis to ensure the faithful translation of genetic information. The maturation of tRNAs and mRNAs is monitored, as is the identity of amino acids attached to tRNAs. Accuracy is further enhanced during the selection of aminoacyl-tRNAs on the ribosome and their base pairing with mRNA. Recent studies have begun to reveal the molecular mechanisms underpinning quality control and go some way to explaining the phenomenal accuracy of translation first observed over three decades ago.     

Proliferating cells express mRNAs with shortened 3' untranslated regions and fewer microRNA target sites

Messenger RNA (mRNA) stability, localization, and translation are largely determined by sequences in the 3' untranslated region (3'UTR). We found a conserved increase in expression of mRNAs terminating at upstream polyadenylation sites after activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3'UTRs, is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues. Forced expression of full-length 3'UTRs conferred reduced protein expression. In some cases the reduction in protein expression could be reversed by deletion of predicted microRNA target sites in the variably included region. Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3'UTR-based regulatory capacity of mRNAs.     

Translational potentiation of messenger RNA with secondary structure in Xenopus

Differential translation of messenger RNA (mRNA) with stable secondary structure in the 5' untranslated leader may contribute to the dramatic changes in protein synthetic patterns that occur during oogenesis and early development. Plasmids that contained the bacterial gene chloramphenicol acetyltransferase and which encoded mRNA with (hpCAT) or without (CAT) a stable hairpin secondary structure in the 5' noncoding region were transcribed in vitro, and the resulting mRNAs were injected into Xenopus oocytes, eggs, and early embryos. During early oogenesis, hpCAT mRNA was translated at less than 3 percent of the efficiency of CAT mRNA. The relative translational potential of hpCAT reached 100 percent in the newly fertilized egg and returned to approximately 3 percent after the midblastula transition.     

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies

A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.     

S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth

The tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5' untranslated region (5'UTR) of messenger RNAs (mRNAs). In response to mitogens, PDCD4 was rapidly phosphorylated on Ser67 by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase SCF(betaTRCP). Expression in cultured cells of a stable PDCD4 mutant that is unable to bind betaTRCP inhibited translation of an mRNA with a structured 5'UTR, resulted in smaller cell size, and slowed down cell cycle progression. We propose that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth.     

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.