A challenge for miRNA: multiple isomiRs in miRNAomics

Accumulating evidence suggests that a single microRNA (miRNA) locus can generate a series of sequences during miRNA maturation process. These multiple sequences, called miRNA variants, or isomiRs, have different lengths and different 5′ and 3′ ends. Some of these isomiRs are detected as varied nucleotides and 3′ additional non-template nucleotides. As physiological miRNA isoforms, they have drawn attention for possible regulatory biological roles. The present work mainly reviews miRNA/isomiR biogenesis, isomiR expression patterns, and functional and evolutionary implications, especially between isomiRs from homologous and clustered miRNA loci. The phenomenon of multiple isomiRs and their biological roles indicates that analysis performed at the miRNA and isomiR levels should be included in miRNA studies. This may enrich and complicate miRNA biogenesis and coding–non-coding RNA regulatory networks.     

Selected isomiR expression profiles via arm switching?

A mature miRNA may be generated from 5p or 3p arm of a hairpin precursor. The selection may be flexible via “arm switching”. However, accumulating evidences suggest that both arms of many pre-miRNAs can yield mature functional miRNAs. Herein, we attempted to compare the isomiR expression profiles between the two arms through analyzing in-house and published small RNA deep sequencing datasets. Although many miR-#-5p and miR-#-3p have been reported as functional miRNAs, fewer miRNA pairs (11 and 6 pairs are collected in tumor and normal cells, respectively) are simultaneously identified as abundant miRNA species. According to isomiR types and dominant isomiR species, miR-#-5p and miR-#-3p show various isomiR expression profiles as well as diverse enrichment levels. IsomiR profiles of non-dominant arm are not well-conserved in 5′ ends as well as isomiR profiles of dominant arm. If both the miR-#-5p and miR-#-3p are abundantly expressed, their isomiR expression profiles are always stable across different samples. Similar to diverse enrichment levels of miR-#-5p and miR-#-3p, the isomiR expression patterns may also be influenced by the phenomenon of “arm switching”. The diverged isomiR expression profiles further enrich the complexity of multiple isomiRs, and complicate the coding-non-coding RNA regulatory network.     

A genome-wide screen for non-template nucleotides and isomiR repertoires in miRNAs indicates dynamic and versatile microRNAome

miRNA variants (termed isomiRs) have been reported as potential functional molecules that may affect miRNA stability or target selection. The aims of the present study were to comprehensively survey and characterize non-template nucleotides (NTNs) and isomiR repertoires in miRNAs. Over 50 % of the NTNs were located in the 3′ ends (also termed 3′ additions), followed by the 5′ ends and adjacent positions to 5′ and 3′ ends. The similar distributions of NTNs and isomiR repertoires might be detected between homologous or clustered miRNAs. miRNA might be stably expressed based on the typical analysis, but its isomiRs might be strongly up- or down-regulated. IsomiRs with novel seed sequences were mainly derived from “seed shifting” events in 5′ isomiRs, NTNs in 5′ ends or in seed sequences. IsomiRs from a miRNA locus or homologous miRNA loci maybe have the same seed sequences, but they would have various enrichment levels and 3′ ends. Interestingly, isomiR species with novel seed sequences via NTNs in the seed region were always stably expressed. These novel seed sequences could lead to novel functional roles through driving the potential novel target mRNAs. Integrated predicted target mRNAs and further microarray validation showed that these isomiRs have versatile biological roles. Collectively, multiple isomiR products and miRNA maturation processes provide opportunities to perform versatile roles in the regulatory network, which further enriches and complicates the regulation of biological processes.     

miRNA异构体：miRNA序列多样性

微RNA（microRNA,miRNA)是一类抑制基因表达的调控分子,在多种生物学进程中扮演重要角色。近来,基于新一代测序技术获得的小RNA测序数据发现,对于某一miRNA来说,它并不是单一的序列,而是由一系列长度/序列及表达不同的异构微RNA (isomicroRNA, isomiRNA/isomiR)所组成。这些isomiR表达多样且序列多样,甚至引入多样的5′端及种子区域。特定miRNA位点在疾病组织中可具有异常的表达模式,现已证实,部分isomiR具有重要的生物学功能。所关注的经典miRNA序列,其实仅是多重isomiR序列中的1条特殊序列,仅从miRNA角度的研究已不足以揭示小RNA的奥秘,全面的研究应同时在miRNA和isomiR中开展,由此可进一步拓宽miRNA的研究思路。本文主要从isomiR的生物学特点、表达及功能等方面进行介绍。     

Adaptive evolution of testis-specific,recently evolved,clustered miRNAs in Drosophila

The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions ∼2–8 (the “seed”), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. In this study, we unexpectedly show that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. We find that multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, we observe that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, our findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis.     

Consistent isomiR expression patterns and 3′ addition events in miRNA gene clusters and families implicate functional and evolutionary relationships

3′ addition events in miRNAs are widely detected and may contribute to miRNA stability, but little is known about details of the events in miRNA gene clusters and families. Here, we performed a comprehensive analysis of isomiR expression patterns and 3′ additions in miRNA gene clusters and families by analyzing high-throughput sequencing dataset. According to dominant modified isomiRs, miRNA members in many miRNA gene clusters and families showed the same 3′ additional non-template nucleotides. Although clustered miRNAs and homologous miRNAs had consistent or inconsistent expression levels, we found many of them showed consistent expression patterns at isomiR levels. These findings revealed similar processing mechanism and 3′ modification event of miRNAs in gene clusters and families through miRNA maturation process. The consistent maturation mechanism may contribute to co-regulate biological processes, and may originate from ancestral miRNA genes through complex duplication history.     

An Exploration of Evolution,Maturation, Expression and Function Relationships in Mir-23～27～24 Cluster

The study aims to explore the potential relationships of evolution, maturation, expression and function between homologous/clustered miRNAs. mir-23∼27∼24 gene cluster, including the two gene clusters (mir-23a and mir-23b) and the three miRNA gene families (mir-23, mir-27 and mir-24), was typically selected as an example. These related miRNAs show similar evolutionary patterns and various expression patterns. Most of them show consistent isomiR expression pattern, and the “switching” phenomenon can be found between different abundant isomiR species. These findings suggest that these sequence or location related miRNAs show the similar miRNA processing and maturation processes, and the robust selection of the most dominant isomiR exists in specific tissues. Functional analysis show that these miRNAs show similar distributions of enriched gene categories, suggesting the close functional prelateships via direct or indirect coordinate regulation in biological processes. The study reveals the close evolutionary, expression and functional relationships between related homologous/clustered miRNAs, which will further enrich miRNA studies and understand direct or indirect interactions between miRNAs.     

Interrogation of rabbit miRNAs and their isomiRs

Rabbit (Oryctolagus cuniculus) is the only lagomorph animal of which the genome has been sequenced. Establishing a rabbit miRNA resource will benefit subsequent functional genomic studies in mammals. We have generated small RNA sequence reads with SOLiD and Solexa platforms to identify rabbit miRNAs, where we identified 464 pre-miRNAs and 886 mature miRNAs. The brain and heart miRNA libraries were used for further in-depth analysis of isomiR distributions. There are several intriguing findings. First, several rabbit pre-miRNAs form highly conserved clusters. Second, there is a preference in selecting one strand as mature miRNA, resulting in an arm selection preference. Third, we analyzed the isomiR expression and validated the expression of isomiR types in different rabbit tissues. Moreover, we further performed additional small RNA libraries and defined miRNAs differentially expressed between brain and heart. We conclude also that isomiR distribution profiles could vary between brain and heart tissues.     

Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5′ and 3′ miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

miRNA–miRNA interaction implicates for potential mutual regulatory pattern

Background

Natural or endogenous sense/antisense miRNAs, located on sense and antisense strands in the same genomic region, respectively, are detected recently. However, little is known about these miRNA pairs, especially for their distributions in different animal species. We herein present systematic analysis of them in human, mouse and rat miRNAs, and their expression patterns based on deep sequencing datasets.

Methods and results

The phenomenon of miRNA–miRNA interaction could be detected in different animal species. The common miRNAs pairs were found across species. These miRNA pairs could form miRNA:miRNA duplex with complete complementary structure, and were prone to be located on specific chromosomes. They might be homologous miRNA genes (especially in human), or clustered in a gene cluster (especially in rat), or simultaneously detected in different genomic regions due to multicopy pre-miRNAs. Remarkably, some miRNA pairs, located in different genomic regions, also showed complementarity as well as endogenous sense/antisense miRNAs. Based on published deep sequencing datasets, one member of miRNA pairs always was abundantly expressed, whereas another was quite rare. Rare common target mRNAs of these miRNA pairs were predicted.

Conclusions

Interaction between miRNAs and significant expression divergence implied complex potential mutual regulatory pattern in the miRNA world. The study would enrich miRNA regulatory network.     

Genetic Analysis of Loop Sequences in the Let-7 Gene Family Reveal a Relationship between Loop Evolution and Multiple IsomiRs

While mature miRNAs have been widely studied, the terminal loop sequences are rarely examined despite regulating both primary and mature miRNA functions. Herein, we attempted to understand the evolutionary pattern of loop sequences by analyzing loops in the let-7 gene family. Compared to the stable miRNA length distributions seen in most metazoans, higher metazoan species exhibit a longer length distribution. Examination of these loop sequence length distributions, in addition to phylogenetic tree construction, implicated loop sequences as the main evolutionary drivers in miRNA genes. Moreover, loops from relevant clustered miRNA gene families showed varying length distributions and higher levels of nucleotide divergence, even between homologous pre-miRNA loops. Furthermore, we found that specific nucleotides were dominantly distributed in the 5′ and 3′ terminal loop ends, which may contribute to the relatively precise cleavage that leads to a stable isomiR expression profile. Overall, this study provides further insight into miRNA processing and maturation and further enriches our understanding of miRNA biogenesis.     

The impact of age,biogenesis, and genomic clustering on Drosophila microRNA evolution

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class.     

Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.