The C‐terminal α–α superhelix of Pat is required for mRNA decapping in metazoa

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N‐terminal sequence, a proline‐rich region, a Mid domain and a C‐terminal domain (Pat‐C). We show that Pat‐C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P‐rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P‐rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P‐rich region. Structural analysis revealed that Pat‐C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat‐C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.     

Exonuclease hDIS3L2 specifies an exosome‐independent 3′‐5′ degradation pathway of human cytoplasmic mRNA

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5′‐3′ exoribonuclease hXRN1 and the 3′‐5′ exoribonucleolytic RNA exosome complex. However, in addition to the exosome‐associated 3′‐5′ exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein—hDIS3L2. Here, we show that hDIS3L2 is an exosome‐independent cytoplasmic mRNA 3′‐5′ exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA‐dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P‐bodies) upon hDIS3L2 depletion, which also increases half‐lives of investigated mRNAs. Consistently, RNA sequencing (RNA‐seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome‐independent effector of cytoplasmic mRNA metabolism.     

Coordinate regulation of histone mRNA metabolism and DNA replication

S phase is characterized by the replication of DNA and assembly of chromatin. This requires the synthesis of large amounts of histone proteins to package the newly replicated DNA. Histone mRNAs are the only mRNAs that do not have polyA tails, ending instead in a conserved stemloop sequence. The stemloop binding protein (SLBP) that binds the 3' end of histone mRNA is cell cycle regulated and SLBP is required in all steps of histone mRNA metabolism. Activation of cyclin E/cdk2 prior to entry into S phase is critical for initiation of DNA replication and histone mRNA accumulation. At the end of S phase SLBP is rapidly degraded as a result of phosphorylation of SLBP by cyclin A/cdk1 and CK2 effectively shutting off histone mRNA biosynthesis. E2F1, which is required for expression of many S-phase genes, is regulated in parallel with SLBP and its degradation also requires a cyclin binding site, suggesting that it may also be regulated by the same pathway. It is likely that activation of cyclin A/cdk1 so helps inhibit both DNA replication and histone mRNA accumulation, marking the end of S phase and entry into G2 phase.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Localization of estrogen receptor‐α and ‐βmRNA in brain areas controlling sexual behavior in Japanese quail

Two estrogen receptors (ERs), denoted ERα and ERβ, have been identified in humans and various animal species, including the Japanese quail. Estrogens play a key role in sexual differentiation and in activation of sexual behavior in Japanese quail. The distribution of ERα in the brain of male and female adult quail has previously been studied using immunohistochemistry, whereas in situ hybridization has been employed to study the distribution of ERβ mRNA in males only. In this article, we used in situ hybridization to study the distribution of mRNAs for both ERα and ERβ in brain areas controlling sexual behavior of Japanese quail. Our results show that both ERα mRNA and ERβ mRNA are localized in areas important for sexual behavior, such as the preoptic area and associated limbic areas, in both males and females. Moreover, we found differences in distribution of mRNA for the two receptors in these areas. The results of this article support previously reported data and provide novel data on localization of ER mRNAs in adult quail brain of both sexes. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

New insights into mRNA decoding — implications for heterologous protein synthesis

The primary structure of a polypeptide can be predicted by translating its mRNA sequence according to the ‘universal’ genetic code. Yet, recent evidence has shown that a number of nonstandard translational events may occur in cells, generating microheterogeneity in the translation product at the amino acid level. Such events can be programmed by sequences within the mRNA, or may just represent nonprogrammed errors that occur during translation as a result of depletion of specific aminoacyl-tRNAs. The potential occurrence of such errors must be considered and steps taken both to identify and eliminate them when expression strategies are being developed for producing recombinant proteins for human therapeutic use.     

Deficiency in mTORC1‐controlled C/EBPβ‐mRNA translation improves metabolic health in mice

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E‐binding proteins (4E‐BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1‐associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPβ) mRNA into the C/EBPβ‐LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E‐BPs is required for suppression of LIP. Intriguingly, mice lacking the cis‐regulatory upstream open reading frame (uORF) in the C/EBPβ‐mRNA, which is required for mTORC1‐stimulated translation into C/EBPβ‐LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPβ‐isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.     

Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders

Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5′ untranslated leaders (5′-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5′-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5′-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5′-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.