C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3′ UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3′ UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3′ UTRs are present on translating ribosomes.     

Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms

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Reinitiation and recycling are distinct processes occurring downstream of translation termination in yeast

The circularisation model of the polysome suggests that ribosome recycling is facilitated by 5'-3' interactions mediated by the cap-binding complex eIF4F and the poly(A)-binding protein, Pab1. Alternatively, downstream of a short upstream open reading frame (uORF) in the 5' untranslated region of a gene, posttermination ribosomes can maintain the competence to (re)initiate translation. Our data show that recycling and reinitiation must be distinct processes in Saccharomyces cerevisiae. The role of the 3'UTR in recycling was assessed by restricting ribosome movement along the mRNA using a poly(G) stretch or the mammalian iron regulatory protein bound to the iron responsive element. We find that although 3'UTR structure can influence translation, the main pathway of ribosome recycling does not depend on scanning-like movement through the 3'UTR. Changes in termination kinetics or disruption of the Pab1-eIF4F interaction do not affect recycling, yet the maintenance of normal in vivo mRNP structure is important to this process. Using bicistronic ACT1-LUC constructs, elongating yeast ribosomes were found to maintain the competence to (re)initiate over only short distances. Thus, as the first ORF to be translated is progressively truncated, reinitiation downstream of an uORF of 105nt is found to be just detectable, and increases markedly in efficiency as uORF length is reduced to 15nt. Experiments using a strain mutated in the Cca1 nucleotidyltransferase suggest that the uORF length-dependence of changes in reinitiation competence is affected by peptide elongation kinetics, but that ORF length per se may also be relevant.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

Tilapia and human CLIC2 structures are highly conserved

Chloride intracellular channels (CLICs) exist in soluble and membrane bound forms. We have determined the crystal structure of soluble Clic2 from the euryhaline teleost fish Oreochromis mossambicus. Structural comparison of tilapia and human CLIC2 with other CLICs shows that these proteins are highly conserved. We have also compared the expression levels of clic2 in selected osmoregulatory organs of tilapia, acclimated to freshwater, seawater and hypersaline water. Structural conservation of vertebrate CLICs implies that they might play conserved roles. Also, tissue-specific responsiveness of clic2 suggests that it might be involved in iono-osmoregulation under extreme conditions in tilapia.     

植物microRNA的生物合成和调控功能

植物microRNA(miRNA)是一类21～24个核苷酸长度的小RNA分子。它的生物合成机制及其对植物生长发育的重要调控作用是人们普遍关注的科学问题和深入探索的研究对象。目前,RNA分子生物学在理论和技术上日趋完善,正在成为一门独立的新兴学科,对生物相关学科的发展产生了重要影响。其中,植物miRNA的生物合成和调控功能是植物小RNA分子生物学的核心问题之一。该文提供植物miRNA领域的最新研究成果,在此基础上对未来的学科发展提出新的建议。     

Evolutionarily conserved and divergent regulatory sequences in the fish rod opsin promoter

Fish have multiple types and subtypes of opsin genes that are expressed in a highly regulated manner in retinal photoreceptor cells. In the rod opsin proximal promoter region (RPPR) of zebrafish (Danio rerio), the BAT 1 regulatory region contains highly conserved OTX (GATTA) and OTX-like (TATTA) sequences that can be recognized by the mammalian cone–rod homeobox (CRX) protein. However, binding of zebrafish crx to the OTX sequence has remained elusive. In contrast to the BAT 1 region, the Ret 1 region, located approximately 20 bp upstream of the BAT 1 region in mammals, is not conserved in zebrafish. In the Ret 1 region, even the core OTX-like sequence (AATTA sequence in mammals) is destructed. We show in this study that a region between Ret 1 and BAT 1 (denoted IRB, Inter-Ret 1-BAT 1) is highly conserved among fish species. Using electrophoretic mobility shift assay (EMSA), we show that zebrafish crx binds to the conserved OTX sequence and that the fish-specific IRB region specifically binds elements present in both retinal and brain nuclear extracts of zebrafish. These results imply that the regulatory mechanisms of opsin gene expression consist not only of evolutionarily conserved but also of divergent machinery among different animal taxa.     

A novel family of plant splicing factors with a Zn knuckle motif: examination of RNA binding and splicing activities

An important group of splicing factors involved in constitutive and alternative splicing contain an arginine/serine (RS)-rich domain. We have previously demonstrated the existence of such factors in plants and report now on a new family of splicing factors (termed the RSZ family) from Arabidopsis thaliana which additionally harbor a Zn knuckle motif similar to the human splicing factor 9G8. Although only around 20 kDa in size, members of this family possess a multi-domain structure. In addition to the N-terminal RNA recognition motif (RRM), a Zn finger motif of the CCHC-type is inserted in an RGG-rich region; all three motifs are known to contribute to RNA binding. The C-terminal domain has a characteristic repeated structure which is very arginine-rich and centered around an SP dipeptide. One member of this family, atRSZp22, has been shown to be a phosphoprotein with properties similar to SR proteins. Furthermore, atRSZp22 was able to complement efficiently splicing deficient mammalian S100 as well as h9G8-depleted extracts. RNA binding assays to selected RNA sequences indicate an RNA binding specificity similar to the human splicing factors 9G8 and SRp20. Taken together, these result show that atRSZp22 is a true plant splicing factor which combines structural and functional features of both h9G8 and hSRp20.     

RNA Ligation and the Origin of tRNA

A straightforward origin of transfer RNA,(tRNA), is difficult to envision because of the apparentlycomplex idiosyncratic interaction between the D-loop and T-loop. Recently, multiple examples of the T-loop structuralmotif have been identified in ribosomal RNA. These examplesshow that the long-range interactions between the T-loop andD-loops seen in tRNA are not an essential part of the motifbut rather are facilitated by it. Thus, the core T-loopstructure could already have existed in a small RNA prior tothe emergence of the tRNA. The tRNA might then have arisenby expansion of an RNA that carried the motif. With thisidea in mind, Di Giulio's earlier hypothesis that tRNAevolved by a simple duplication or ligation of a minihelixRNA was re-examined. It is shown that an essentially moderntRNA structure can in fact be generated by the ligation oftwo 38-nucleotide RNA minihelices of appropriate sequence.Although rare, such sequences occur with sufficientfrequency, (1 in 3 × 10⁷), that they could be found in astandard in vitro RNA selection experiment. Theresults demonstrate that a series of RNA duplications, aspreviously proposed, can in principal account for the originof tRNA. More generally, the results point out that RNAligation can be a powerful driving force for increasedcomplexity in the RNA World.     

One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences,structures and functions

The eightfold (betaalpha) barrel structure, first observed in triose-phosphate isomerase, occurs ubiquitously in nature. It is nearly always an enzyme and most often involved in molecular or energy metabolism within the cell. In this review we bring together data on the sequence, structure and function of the proteins known to adopt this fold. We highlight the sequence and functional diversity in the 21 homologous superfamilies, which include 76 different sequence families. In many structures, the barrels are "mixed and matched" with other domains generating additional variety. Global and local structure-based alignments are used to explore the distribution of the associated functional residues on this common structural scaffold. Many of the substrates/co-factors include a phosphate moiety, which is usually but not always bound towards the C-terminal end of the sequence. Some, but not all, of these structures, exhibit a structurally conserved "phosphate binding motif". In contrast metal-ligating residues and catalytic residues are distributed along the sequence. However, we also found striking structural superposition of some of these residues. Lastly we consider the possible evolutionary relationships between these proteins, whose sequences are so diverse that even the most powerful approaches find few relationships, yet whose active sites all cluster at one end of the barrel. This extreme example of the "one fold-many functions" paradigm illustrates the difficulty of assigning function through a structural genomics approach for some folds.     

Human, Drosophila, and C.elegans TDP43: nucleic acid binding properties and splicing regulatory function

TAR DNA binding protein (TDP43), a highly conserved heterogeneous nuclear ribonucleoprotein, was found to down-regulate splicing of the exon 9 cystic fibrosis transmembrane conductance regulator (CFTR) through specific binding to a UG-rich polymorphic region upstream of the 3' splice site. Despite the emergence of new information regarding the protein's nuclear localization and splicing regulatory activity, TDP43's role in cells remains elusive. To investigate the function of human TDP43 and its homologues, we cloned and characterized the proteins from Drosophila melanogaster and Caenorhabditis elegans. The proteins from human, fly, and worm show striking similarities in their nucleic acid binding specificity. We found that residues at two different positions, which show a strong conservation among TDP43 family members, are linked to the tight recognition of the target sequence. Our three-dimensional model of TDP43 in complex with a (UG)(m) sequence predicts that these residues make amino acid side-chain to base contacts. Moreover, our results suggest that Drosophila TDP43 is comparable to human TDP43 in regulating exon splicing. On the other hand, C.elegans TDP43 has no effect on exon recognition. TDP43 from C.elegans lacks the glycine-rich domain found at the carboxy terminus of the other two homologues. Mutants of human and fly TDP43 devoid of the C-terminal domain are likewise unable to affect splicing. Our studies suggest that the glycine-rich domain is essential for splicing regulation by human and fly TDP43.