Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

Prediction of m6A and m5C at single-molecule resolution reveals a transcriptome-wide co-occurrence of RNA modifications.

The epitranscriptome embodies many new and largely unexplored functions of RNA. A significant roadblock hindering progress in epitranscriptomics is the identification of more than one modification in individual transcript molecules. We address this with CHEUI (CH3 (methylation) Estimation Using Ionic current). CHEUI predicts N6-methyladenosine (m6A) and 5-methylcytosine (m5C) in individual molecules from the same sample, the stoichiometry at transcript reference sites, and differential methylation between any two conditions. CHEUI processes observed and expected nanopore direct RNA sequencing signals to achieve high single-molecule, transcript-site, and stoichiometry accuracies in multiple tests using synthetic RNA standards and cell line data. CHEUI's capability to identify two modification types in the same sample reveals a co-occurrence of m6A and m5C in individual mRNAs in cell line and tissue transcriptomes. CHEUI provides new avenues to discover and study the function of the epitranscriptome.

From factors to mechanisms: translation and translational control in eukaryotes.

Biochemical and genetic studies are revealing a network of interactions between eukaryotic translation initiation factors, further refining or redefining perceptions of their function. The notion of translated mRNA as a 'closed-loop' has gained support from the identification of physical and functional interactions between the two mRNA ends and their associated factors. Translational control mechanisms are beginning to unravel in sufficient detail to pinpoint the affected step in the initiation pathway.

Yeast 18S rRNA dimethylase Dim1p: a quality control mechanism in ribosome synthesis?

One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3' end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2 strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate of dim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGK promoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

The tissue-specific RNA-binding protein COLBP is differentially regulated during myogenesis.

Cytochrome c oxidase L-form transcript-binding protein (COLBP) activity parallels the tissue-specific mRNA expression of bovine cytochrome c oxidase liver isopeptides. A similar RNA-binding activity is found in human myoblast and liver Hep G2 cell homogenates. Human COLBP activity, however, is not present in myotubes or adult skeletal muscle. It is proposed that COLBP down-regulation during muscle cell differentiation may underlie oxidase isoform switching during myogenesis.

Developmental regulation of tissue-specific isoforms of subunit VIa of beef cytochrome c oxidase.

The switching of the subunit VIa isoforms of cytochrome c oxidase has been followed in heart tissue during bovine development both by transcript levels and in terms of the incorporation of L- (liver) and H- (heart) polypeptides into mitochondria. In early fetuses, e.g., 60-days development, there are high levels of VIaL transcript and high levels of the VIaL polypeptide incorporated into mitochondria. In late fetuses (after 200 days), the levels of VIaL transcript are still high, with less but still significant amounts of VIaL polypeptide present in comparison to adult heart in which the amount of this isoform is negligible. As the proportion of VIaL transcript is reduced, the proportion of VIaH transcript increases along with the amount of the VIaH isoform in mitochondria. These data indicate isoform switching during late fetal development. The presence of COLBP (cytochrome oxidase liver isoform binding protein) (Preiss, T. and Lightowlers, R.N. (1993) J. Biol. Chem. 268, 10659-10667) was examined at different developmental stages. COLBP binding activity was observed in hearts of late fetuses but not found in adult heart tissue, providing a correlation between the presence of this factor and the presence of the VIaL polypeptide in mitochondria.

The mRNA-binding protein COLBP is glutamate dehydrogenase.

Expression of the liver-type isopeptides of cytochrome c oxidase is regulated post-transcriptionally. An RNA-binding activity has been found in only those cells where the liver-type subunits are detected. This binding protein, termed COLBP, recognises sequences or structures within the 3'-untranslated regions of transcripts encoding these liver-type isopeptides and has been implicated in the modulation of mRNA expression. We now show by subcellular fractionation, immunocompetition, UV-crosslinking and shift-Western studies that the metabolic enzyme glutamate dehydrogenase, previously reported as being able to bind RNA, is the cytochrome c oxidase transcript-binding protein, COLBP.

Expression and functional analysis of steroid receptor fragments secreted from Staphylococcus aureus.

Fragments of the DNA-binding domain of the rat glucocorticoid receptor (rGR) and the human estrogen receptor (hER) were expressed in Staphylococcus aureus as a chimeric fusion to protein A by using a modified expression vector with an artificial factor X-cleavage site. The secreted product was isolated by hydrophobic chromatography on Phenyl-Sepharose and purified on DNA-cellulose or by anion-exchange chromatography. After cleavage of the protein A moiety, the purified rGR DNA-binding domain from amino acids 406 to 523 (rGR406-523), binds specifically to a glucocorticoid responsive element as a homodimer but cannot form heterodimers with the DNA-binding domain of the hER. Amino acids 510 to 523 following the zinc finger region, as well as free sulfhydryl-groups are necessary for DNA-binding, which is more efficient when the tripeptide Gly-Gly-Cys is added to the carboxy terminal end. Despite its specific interaction with DNA, rGR406-523 does not activate transcription from the MMTV promoter in a cell-free system that efficiently responds to addition of native GR, suggesting that regions essential for transcriptional activation in vitro are located outside of the DNA-binding domain.

Two independent pathways for transcription from the MMTV promoter.

The influence of progesterone receptor (PR) and glucocorticoid receptor (GR) on transcription from the mouse mammary tumour virus (MMTV) promoter was analyzed using cell-free transcription of DNA templates with a G-free cassette. Preincubation of the templates with either PR or GR stimulates the rate of transcription initiation 10-50 fold, whereas the recombinant DNA binding domain of GR is inactive. Mutations that inactivate the nuclear factor I (NFI) binding site, or NFI depletion of the nuclear extract, decrease basal transcription without influencing receptor-dependent induction. Recombinant NFI, but not its DNA-binding domain, restores efficient basal transcription of the depleted extract. Recombinant OTF1 or OTF2, but not the POU domain of OTF1, enhance MMTV transcription independently of NF1. In agreement with this finding, NFI and OTF1 do not cooperate, but rather compete for binding to the wild type MMTV promoter, though they have the potential to bind simultaneously to properly oriented sites. Our results imply the existence of two independent pathways for MMTV transcription: one initiated by NFI and the other dependent on octamer transcription factors. Only the second pathway is stimulated by steroid hormone receptors in vitro.