The increase of bodyplan complexity in early bilaterian evolution is correlates with the advent and diversification of microRNAs. These small RNAs guide animal development by regulating temporal transitions in gene expression involved in cell fate choices and transitions between pluripotency and differentiation. One of the two known microRNAs whose origins date back before the bilaterian ancestor is mir-100. In Bilateria, it appears stably associated in polycistronic transcripts with let-7 and mir-125, two key regulators of development. In vertebrates, these three microRNA families have expanded to form a complex system of developmental regulators. In this contribution, we disentangle the evolutionary history of the let-7 locus, which was restructured independently in nematodes, platyhelminths, and deuterostomes. The foundation of a second let-7 locus in the common ancestor of vertebrates and urochordates predates the vertebrate-specific genome duplications, which then caused a rapid expansion of the let-7 family.
Evolution and function of the extended miR-2 microRNA family
MicroRNAs are essential post-transcriptional regulators. Many animal microRNAs are clustered in the genome, and it has been shown that clustered microRNAs may be transcribed as a single transcript. Polycistronic microRNAs are often members of the same family, suggesting a role of tandem duplication in the emergence of clusters. The mir-2 microRNA family is the largest in Drosophila melanogaster, with 8 members that are mostly clustered in the genome. Previous studies suggest that the copy number and genomic distribution of mir-2 family members has been subject to significant change during evolution. The effects of such changes on their function are still unknown. Here we study the evolution of function in the mir-2 family. Our analyses show that, in spite of the change in number and organization among invertebrates, most mir-2 loci produce very similar mature microRNA products. Multiple mature miR-2 sequences are predicted to target genes involved in neural development in Drosophila. These targeting properties are conserved in the distant species Caenorhabditis elegans. Duplication followed by functional diversification is frequent during protein-coding gene evolution. However, our results suggest that the production of microRNA clusters by gene duplication rarely involves functional changes. This pattern of functional redundancy among clustered paralogous microRNAs reflects birth-and-death evolutionary dynamics. However, we identified a small number of mir-2 sequences in Drosophila that may have undergone functional shifts associated with genomic rearrangements. Therefore, redundancy in microRNA families may facilitate the acquisition of novel functional features.
Are all the miRBase-registered microRNAs true?: A structure- and expression-based re-examination in plants
In this survey, we did a large-scale re-examination of the currently registered plant microRNAs (miRNAs) in miRBase (release 17), which were annotated based on the already established criteria. Huge public small RNA (sRNA) high-throughput sequencing (HTS) data sets were employed to interrogate the accuracy of the miRBase registries based on the secondary structures of the miRNA precursors and the expression levels of the miRNAs and the miRNA*s. Our results raised the caveat that the current miRNA lists in miRBase should be carefully refined, and more strict criteria should be implemented for new miRNA registration. Through this work, we proposed a structure- and expression-based strategy to validate a set of defined miRNA genes, or even to annotate novel ones based on currently available sRNA HTS data sets. We also hope to inspire further research efforts on the manual refinement of the current miRNA gene lists.
A structural module in RNase P expands the variety of RNA kinks
RNA structures are built from recurrent modules that can be identified by structural and comparative sequence analysis. In order to assemble sets of helices in compact architectures, modules that introduce bends and kinks are necessary. Among such modules, kink-turns form an important family that presents sequence and structural characteristics. Here, we describe an internal loop in the bacterial type A RNase P RNA that sets helices bound at the junctions exactly in the same relative positions as in kink-turns but without the structural signatures typical of kink-turns. Our work suggests that identifying a structural module in a subset of RNA sequences constitutes a strategy to identify distinct sequential motifs sharing common structural characteristics.
Microtubule-dependent mRNA transport in the model microorganism Ustilago maydis
Microtubule-dependent trafficking is essential in moving mRNAs over long distances. This transport mechanism regulates important cellular events such as determining polarity and local protein secretion. Key examples are developmental and neuronal processes studied in Drosophila melanogaster, Xenopus laevis as well as in mammalian cells. A simple eukaryotic system to uncover basic mechanisms was missing. Fungal models are generally well suited for this purpose, since transgenic strains can be generated easily by homologous recombination allowing in vivo studies at native expression levels. Substantial advances in understanding Ustilago maydis showed that this fungus fulfils important criteria to serve as model for microtubule-dependent mRNA trafficking. Here, we summarize progress focusing on target mRNAs, RNA localization elements, RNA-binding proteins, mRNPs, molecular motors and microtubule organization. This serves as the basis to discuss the novel mechanism of mRNP hitchhiking on endosomes as well as an unexpected link to unconventional secretion with its implications for applied sciences.
microRNAs (miRNAs), defined as 21–24 nucleotide non-coding RNAs, are important regulators of gene expression. Initially, the functions of miRNAs were recognized as post-transcriptional regulators on mRNAs that result in mRNA degradation and/or translational repression. It is becoming evident that miRNAs are not only restricted to function in the cytoplasm, they can also regulate gene expression in other cellular compartments by a spectrum of targeting mechanisms via coding regions, 5′ and 3′untransalated regions (UTRs), promoters, and gene termini. In this point-of-view, we will specifically focus on the nuclear functions of miRNAs and discuss examples of miRNA-directed transcriptional gene regulation identified in recent years.
Non-coding transcription characterization and annotation: A guide and web resource for non-coding RNA databases
Large-scale transcriptome projects have shown that the number of RNA transcripts not coding for proteins (non-coding RNAs) is much larger than previously recognized. High-throughput technologies, coupled with bioinformatics approaches, have produced increasing amounts of data, highlighting the role of non-coding RNAs (ncRNAs) in biological processes. Data generated by these studies include diverse non-coding RNA classes from organisms of different kingdoms, which were obtained using different experimental and computational assays. This has led to a rapid increase of specialized RNA databases. The fast growth in the number of available databases makes integration of stored information a difficult task. We present here NRDR, a Non-coding RNA Databases Resource for information retrieval on ncRNA databases (www.ncrnadatabases.org). We performed a survey of 102 public databases on ncRNAs and we have introduced four categorizations to classify these databases and to help researchers quickly search and find the information they need: RNA family, information source, information content and available search mechanisms. NRDR is a useful databases searching tool that will facilitate research on ncRNAs.
Pathogenesis-related mutations in the T-loops of human mitochondrial tRNAs affect 3′ end processing and tRNA structure
Numerous mutations in the mitochondrial genome are associated with maternally transmitted diseases and syndromes that affect muscle and other high energy-demand tissues. The mitochondrial genome encodes 13 polypeptides, 2 rRNAs and 22 interspersed tRNAs via long bidirectional polycistronic primary transcripts, requiring precise excision of the tRNAs. Despite making up only ~10% of the mitochondrial genome, tRNA genes harbor most of the pathogenesis-related mutations. tRNase Z endonucleolytically removes the pre-tRNA 3′ trailer. The flexible arm of tRNase Z recognizes and binds the elbow (including the T-loop) of pre-tRNA. Pathogenesis-related T-loop mutations in mitochondrial tRNAs could thus affect tRNA structure, reduce tRNase Z binding and 3′ processing, and consequently slow mitochondrial protein synthesis. Here we inspect the effects of pathogenesis-related mutations in the T-loops of mitochondrial tRNAs on pre-tRNA structure and tRNase Z processing. Increases in KM arising from 59A > G substitutions in mitochondrial tRNAGly and tRNAIle accompany changes in T-loop structure, suggesting impaired substrate binding to enzyme.
MicroRNAs (miRNA) are small noncoding RNAs that play important roles in cell development, differentiation and apoptosis. Recent studies showed that transmembrane protein 59 (TMEM59) affects neural stem cell (NSC) differentiation and β-amyloid precursor protein (APP) glycosylation. In this study, we predicted that microRNA-351 (miR-351) could target TMEM59 and demonstrated that miR-351 negatively regulates TMEM59 expression in different cell types. Moreover, we found that miR-351 overexpression could lead to morphological change in the mouse NSC cell line C17.2, indicating an important role of miR-351 in the regulation of differentiation. Our investigations focused on the functions of miR-351 in the nervous system and the posttranscriptional regulation of TMEM59 by microRNA. These original findings provide promising grounds for future in-depth research into the functions of miR-351 and TMEM59 in nervous system development.
A novel hypoxic stress-responsive long non-coding RNA transcribed by RNA polymerase III in Arabidopsis
Recently, a large number of non-coding RNAs (ncRNAs) have been found in a wide variety of organisms, but their biological functions are poorly understood, except for several tiny RNAs. To identify novel ncRNAs with essential functions in flowering plants, we focused attention on RNA polymerase III (Pol III) and its transcriptional activity, because most Pol III-transcribed RNAs contribute to key processes relating to cell activities, and have highly conserved promoter elements: upstream sequence elements, a TATA-like sequence, and a poly(T) stretch as a transcription terminator. After in silico prediction from the Arabidopsis genome, 20 novel ncRNAs candidates were obtained. AtR8 RNA (approx. 260 nt) and AtR18 RNA (approx. 160 nt) were identified by efficient in vitro transcription by Pol III in tobacco nuclear extracts. AtR8 RNA was conserved among six additional taxa of Brassicaceae, and the secondary structure of the RNA was also conserved among the orthologs. Abundant accumulation of AtR8 RNA was observed in the plant roots and cytosol of cultured cells. The RNA was not processed into a smaller fragment and no short open reading frame was included. Remarkably, expression of the AtR8 RNA responded negatively to hypoxic stress, and this regulation evidently differed from that of U6 snRNA.
Intergenic Polycomb target sites are dynamically marked by non-coding transcription during lineage commitment
Non-coding (nc) RNAs are involved both in recruitment of vertebrate Polycomb (PcG) proteins to chromatin, and in activation of PcG target genes. Here we investigate dynamic changes in the relationship between ncRNA transcription and recruitment of PcG proteins to chromatin during differentiation. Profiling of purified cell populations from different stages of a defined murine in vitro neural differentiation system shows that over 50% of regulated intergenic non-coding transcripts precisely correspond to PcG target sites. We designate these PcG recruiting elements as Transcribed Intergenic Polycomb (TIP) sites. The relationship between TIP transcription and PcG recruitment switches dynamically during differentiation between different states, in which transcription and PcG recruitment exclude each other, or in which both are present. Reporter assays show that transcribed TIP sites can repress a flanking gene. Knockdown experiments demonstrate that TIP ncRNAs are themselves required for repression of target genes both in cis and in trans. We propose that TIP transcription may ensure coordinated regulation of gene networks via dynamic switching and recruitment of PcG proteins both in cis and in trans during lineage commitment.
A chloroplastic RNA ligase activity analogous to the bacterial and archaeal 2´–5′ RNA ligase
Bacteria and archaea contain a 2’-5′ RNA ligase that seals in vitro 2’,3′-cyclic phosphodiester and 5′-hydroxyl RNA termini, generating a 2’,5′-phosphodiester bond. In our search for an RNA ligase able to circularize the monomeric linear replication intermediates of viroids belonging to the family Avsunviroidae, which replicate in the chloroplast, we have identified in spinach (Spinacea oleracea L.) chloroplasts a new RNA ligase activity whose properties resemble those of the bacterial and archaeal 2’-5′ RNA ligase. The spinach chloroplastic RNA ligase recognizes the 5′-hydroxyl and 2’,3′-cyclic phosphodiester termini of Avocado sunblotch viroid and Eggplant latent viroid RNAs produced by hammerhead-mediated self-cleavage, yielding circular products linked through an atypical, most likely 2’,5′-phosphodiester, bond. The enzyme neither requires divalent cations as cofactors, nor NTPs as substrate. The reaction apparently reaches equilibrium at a low ratio between the final circular product and the linear initial substrate. Even if its involvement in viroid replication seems unlikely, the identification of a 2’-5′ RNA ligase activity in higher plant chloroplasts, with properties very similar to an analogous enzyme widely distributed in bacterial and archaeal proteomes, is intriguing and suggests an important biological role so far unknown.
Tri-snRNP-associated proteins interact with subunits of the TRAMP and nuclear exosome complexes, linking RNA decay and pre-mRNA splicing
Nuclear RNA decay factors are involved in many different pathways including rRNA processing, snRNA and snoRNA biogenesis, pre-mRNA processing, and the rapid decay of cryptic intergenic transcripts. In contrast to its yeast counterpart, the mammalian nuclear decay machinery is largely uncharacterized. Here we report interactions of several putative components of the human nuclear RNA decay machinery, including the TRAMP complex protein Mtr4 and the nuclear exosome constituents PM/Scl-100 and PM/Scl-75, with components of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing. The tri-snRNP component Prp31 interacts indirectly with Mtr4 and PM/Scl-100 in a manner that is dependent on the phosphorylation sites in the middle of the protein, while Prp3 and Prp4 interact with the nuclear decay complex independent of Prp31. Together our results suggest recruitment of the nuclear decay machinery to the spliceosome to ensure production of properly spliced mRNA.
The ordered processing of intervening sequences in 23S rRNA of Rhodobacter sphaeroides requires RNase J
The essential processing of ribosomal rRNA precursors requires concerted and sequential cleavages by different endo- and exoribonucleases. Despite long lasting investigations of these processes the exact order of steps remained elusive. Many bacteria perform additional rRNA processing steps by removing intervening sequences within the 23S rRNA. This leads to disintegration of the 23S rRNA and discontinuously assembled fragments within the ribosomes. The maturation of these fragments also requires successive cleavage events by different RNases. Our study reveals that the 5′-to-3′ exoribonuclease RNase J is responsible for the final 5′-end maturation of all three 23S rRNA fragments in the α-proteobacterium Rhodobacter sphaeroides. Additionally the results show that 5′- and 3′-processing steps are closely coupled: mature 5′-ends are a strict prerequisite for the final 3′-trimming of the 23S rRNA fragments.
Modeling SNP mediated differential targeting of homologous 3′UTR by microRNA
We had previously proposed that the post-transcriptional regulation through microRNA as a mechanism for incomplete penetrance and variable expressivity, leads to lack of correlation between genotype and phenotype. Here we report the validation of miRNA-target interactions we predicted earlier and demonstrate the regulation of endogenous JAG1 by hsa-miR-214 and hsa-miR-124, and TGFBR2 by hsa-miR-34b*, through luciferase activity of reporter constructs and also the expression levels of the endogenous genes. Using these targets, we have modeled the diploid state for miRNA target site with heterozygosity for the SNP and demonstrate the differential targeting of an otherwise identical 3′UTR. We show that SNP rs8708 (A > G) at the target site of hsa-miR-214 can relieve the repression while an SNP rs11466532 (C > T) enhances the repression of reporter expression by hsa-miR-34b*. We discuss the results in the light of its implications in the context of penetrance of dominant mutations in miRNA targeted genes, using JAG1 as an example. These observations imply that disease causing mutations in JAG1 linked to the SNP rs8708G will be poorly targeted by hsa-miR-214 when present against a normal allele of JAG1 with rs8708A and will show penetrance of JAG1 mutations as Alagille syndrome, while mutant JAG1 linked to rs8708A against rs8708G on the normal allele will show either no disease or much attenuated symptoms and hence exhibit incomplete penetrance.