NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

LINC00116 codes for a mitochondrial peptide linking respiration and lipid metabolism.

Genes coding for small peptides have been frequently misannotated as long noncoding RNA (lncRNA) genes. Here we have demonstrated that one such transcript is translated into a 56-amino-acid-long peptide conserved in chordates, corroborating the work published while this manuscript was under review. The Mtln peptide could be detected in mitochondria of mouse cell lines and tissues. In line with its mitochondrial localization, lack of the Mtln decreases the activity of mitochondrial respiratory chain complex I. Unlike the integral components and assembly factors of NADH:ubiquinone oxidoreductase, Mtln does not alter its enzymatic activity directly. Interaction of Mtln with NADH-dependent cytochrome b5 reductase stimulates complex I functioning most likely by providing a favorable lipid composition of the membrane. Study of Mtln illuminates the importance of small peptides, whose genes might frequently be misannotated as lncRNAs, for the control of vitally important cellular processes.

eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop.

Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3'-untranslated regions (3'-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the functional consequences of PCBP2 binding to 5'-UTRs. Here we show that the PCBP2 isoform f interacts with the 5'-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I translation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another's expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein.

Structural and evolutionary insights into ribosomal RNA methylation.

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. Identification of all enzymes responsible for rRNA methylation, as well as mapping of all modified rRNA residues, is now complete for a number of model species, such as Escherichia coli and Saccharomyces cerevisiae. Recent high-resolution structures of bacterial ribosomes provided the first direct visualization of methylated nucleotides. The structures of ribosomes from various organisms and organelles have also lately become available, enabling comparative structure-based analysis of rRNA methylation sites in various taxonomic groups. In addition to the conserved core of modified residues in ribosomes from the majority of studied organisms, structural analysis points to the functional roles of some of the rRNA methylations, which are discussed in this Review in an evolutionary context.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Mining for Small Translated ORFs.

Peptides encoded by short open reading frames (sORFs) are usually defined as peptides ≤100 aa long. Usually sORFs were ignored by automatic genome annotation programs due to the high probability of false discovery. However, improved computational tools along with a high-throughput RIBO-seq approach identified a myriad of translated sORFs. Their importance becomes evident as we are gaining experimental validation of their diverse cellular functions. This Review examines various computational and experimental approaches of sORFs identification as well as provides the summary of our current knowledge of their functional roles in cells.

Williams-Beuren Syndrome Related Methyltransferase WBSCR27: From Structure to Possible Function.

Williams-Beuren syndrome (WBS) is a genetic disorder associated with the hemizygous deletion of several genes in chromosome 7, encoding 26 proteins. Malfunction of these proteins induce multisystemic failure in an organism. While biological functions of most proteins are more or less established, the one of methyltransferase WBSCR27 remains elusive. To find the substrate of methylation catalyzed by WBSCR27 we constructed mouse cell lines with a Wbscr27 gene knockout and studied the obtained cells using several molecular biology and mass spectrometry techniques. We attempted to pinpoint the methylation target among the RNAs and proteins, but in all cases neither a direct substrate has been identified nor the protein partners have been detected. To reveal the nature of the putative methylation substrate we determined the solution structure and studied the conformational dynamic properties of WBSCR27 in apo state and in complex with S-adenosyl-L-homocysteine (SAH). The protein core was found to form a canonical Rossman fold common for Class I methyltransferases. N-terminus of the protein and the ß6-ß7 loop were disordered in apo-form, but binding of SAH induced the transition of these fragments to a well-formed substrate binding site. Analyzing the structure of this binding site allows us to suggest potential substrates of WBSCR27 methylation to be probed in further research.

N6-Methylated Adenosine in RNA: From Bacteria to Humans.

N6-methyladenosine (m(6)A) is ubiquitously present in the RNA of living organisms from Escherichia coli to humans. Methyltransferases that catalyze adenosine methylation are drastically different in specificity from modification of single residues in bacterial ribosomal or transfer RNA to modification of thousands of residues spread among eukaryotic mRNA. Interactions that are formed by m(6)A residues range from RNA-RNA tertiary contacts to RNA-protein recognition. Consequences of the modification loss might vary from nearly negligible to complete reprogramming of regulatory pathways and lethality. In this review, we summarized current knowledge on enzymes that introduce m(6)A modification, ways to detect m(6)A presence in RNA and the functional role of this modification everywhere it is present, from bacteria to humans.

Strong effect of copper(II) coordination on antiproliferative activity of thiosemicarbazone-piperazine and thiosemicarbazone-morpholine hybrids.

In this study, 2-formylpyridine thiosemicarbazones and three different heterocyclic pharmacophores were combined to prepare thiosemicarbazone­piperazine mPip-FTSC (HL1) and mPip-dm-FTSC (HL2), thiosemicarbazone­morpholine Morph-FTSC (HL3) and Morph-dm-FTSC (HL4), thiosemicarbazone­methylpyrrole-2-carboxylate hybrids mPyrr-FTSC (HL5) and mPyrr-dm-FTSC (HL6) as well as their copper(II) complexes [CuCl(mPipH-FTSC-H)]Cl (1 + H)Cl, [CuCl(mPipH-dm-FTSC-H)]Cl (2 + H)Cl, [CuCl(Morph-FTSC-H)] (3), [CuCl(Morph-dm-FTSC-H)] (4), [CuCl(mPyrr-FTSC-H)(H2O)] (5) and [CuCl(mPyrr-dm-FTSC-H)(H2O)] (6). The substances were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy (HL1­HL6), ESI mass spectrometry, IR and UV­vis spectroscopy and single crystal X-ray diffraction (1­5). All compounds were prepared in an effort to generate potential antitumor agents with an improved therapeutic index. In addition, the effect of structural alterations with organic hybrids on aqueous solubility and copper(II) coordination ability was investigated. Complexation of ligands HL2 and HL4 with copper(II) was studied in aqueous solution by pH-potentiometry, UV­vis spectrophotometry and EPR spectroscopy. Proton dissociation processes of HL2 and HL4 were also characterized in detail and microscopic constants for the Z/E isomers were determined. While the hybrids HL5, HL6 and their copper(II) complexes 5 and 6 proved to be insoluble in aqueous solution, precluding antiproliferative activity studies, the thiosemicarbazone­piperazine and thiosemicarbazone­morpholine hybrids HL1­HL4, as well as copper(II) complexes 1­4 were soluble in water enabling cytotoxicity assays. Interestingly, the metal-free hybrids showed very low or even a lack of cytotoxicity (IC50 values > 300 µM) in two human cancer cell lines HeLa (cervical carcinoma) and A549 (alveolar basal adenocarcinoma), whereas their copper(II) complexes were cytotoxic showing IC50 values from 25.5 to 65.1 µM and 42.8 to 208.0 µM, respectively in the same human cancer cell lines after 48 h of incubation. However, the most sensitive for HL4 and complexes 1­4 proved to be the human cancer cell line LS174 (colon carcinoma) as indicated by the calculated IC50 values varying from 13.1 to 17.5 µM.