Paralogous miRNA Research Articles

Mutation Screening in the miR-183/96/182 Cluster in Patients With Inherited Retinal Dystrophy.

Inherited retinal dystrophy (IRD) is a heterogenous blinding eye disease and affects more than 200,000 Americans and millions worldwide. By far, 270 protein-coding genes have been identified to cause IRD when defective. However, only one microRNA (miRNA), miR-204, has been reported to be responsible for IRD when a point-mutation occurs in its seed sequence. Previously, we identified that a conserved, polycistronic, paralogous miRNA cluster, the miR-183/96/182 cluster, is highly specifically expressed in all photoreceptors and other sensory organs; inactivation of this cluster in mice resulted in syndromic IRD with multi-sensory defects. We hypothesized that mutations in the miR-183/96/182 cluster in human cause IRD. To test this hypothesis, we perform mutation screening in the pre-miR-183, -96, -182 in >1000 peripheral blood DNA samples of patients with various forms of IRD. We identified six sequence variants, three in pre-miR-182 and three in pre-miR-96. These variants are in the pre-miRNA-182 or -96, but not in the mature miRNAs, and are unlikely to be the cause of the IRD in these patients. In spite of this, the nature and location of these sequence variants in the pre-miRNAs suggest that some may have impact on the biogenesis and maturation of miR-182 or miR-96 and potential roles in the susceptibility to diseases. Although reporting on negative results so far, our study established a system for mutation screening in the miR-183/96/182 cluster in human for a continued effort to unravel and provides deeper insight into the potential roles of miR-183/96/182 cluster in human diseases.

Three paralogous clusters of the miR-17~92 family of microRNAs restrain IL-12-mediated immune defense.

MicroRNAs (miRNAs) have been widely implicated in immune regulation, but evidence for the coordinated function of paralogous miRNA clusters remains scarce. Here, by using genetically modified mice with individual or combined cluster deficiencies, we found that three paralogous clusters of the miR-17~92 family of miRNAs collectively suppressed IL-12 production in macrophages. Accordingly, miR-17~92 family miRNAs deficiencies resulted in heightened production of IL-12 and thus enhanced the host defense against intracellular pathogen Listeria monocytogenes in vivo. Mechanistically, different members of the miR-17~92 family of miRNAs acted on a common target, PTEN, to inhibit IL-12 expression by modulating the PI3K-Akt-GSK3 pathway. In addition, the expression of miR-17~92 family miRNAs was collectively inhibited by the transcription factor RBP-J, and RBP-J-associated macrophage functional defects were genetically rescued by deleting three clusters of miR-17~92 family miRNAs on a RBP-J null background. Thus, our results illustrated key roles of three clusters of miR-17~92 family miRNAs in cooperatively controlling IL-12-mediated immune responses and identified miR-17~92 family miRNAs as functional targets of RBP-J in macrophages.

MicroRNA duplication accelerates the recruitment of new targets during vertebrate evolution.

The repertoire of miRNAs has considerably expanded during metazoan evolution, and duplication is an important mechanism for generating new functional miRNAs. However, relatively little is known about the functional divergence between paralogous miRNAs and the possible coevolution between duplicated miRNAs and the genomic contexts. By systematically examining small RNA expression profiles across various human tissues and interrogating the publicly available miRNA:mRNA pairing chimeras, we found that changes in expression patterns and targeting preferences are widespread for duplicated miRNAs in vertebrates. Both the empirical interactions and target predictions suggest that evolutionarily conserved homo-seed duplicated miRNAs pair with significantly higher numbers of target sites compared to the single-copy miRNAs. Our birth-and-death evolutionary analysis revealed that the new target sites of miRNAs experienced frequent gains and losses during function development. Our results suggest that a newly emerged target site has a higher probability to be functional and maintained by natural selection if it is paired to a seed shared by multiple paralogous miRNAs rather than being paired to a single-copy miRNA. We experimentally verified the divergence in target repression between two paralogous miRNAs by transfecting let-7a and let-7b mimics into kidney-derived cell lines of four mammalian species and measuring the resulting transcriptome alterations by extensive high-throughput sequencing. Our results also suggest that the gains and losses of let-7 target sites might be associated with the evolution of repressiveness of let-7 across mammalian species.

The role of miR-130a in cancer.

MicroRNAs (miRs) are short and highly conserved non-coding RNAs molecules consisting of 18-25 nucleotides that regulate gene expression at post-transcriptional level by direct binding to complementary binding sites within the 3'untranslated region (3'UTR) of target mRNAs. New evidences have demonstrated that miRNAs play an important role in diverse physiological processes, including regulating cell growth, apoptosis, metastasis, drug resistance, and invasion. In chromosomes 11 and 22 of the miR-130 family, paralogous miRNA sequences, miR-130a and miR-130b are situated, respectively. MiR-130a has participated in different pathogenesis, including hepatocellular carcinoma, cervical cancer, ovarian cancer, glioblastoma, prostate carcinoma, leukemia, etc. Most important of all, more and more evidences indicate that miR-130a is associated with drug resistance and acts as an intermediate in PI3K/Akt/PTEN/mTOR, Wnt/β-catenin and NF-kB/PTEN drug resistance signaling pathways. Drug resistance has emerged as a major obstacle to successful treatment of cancer nowadays and in this review, we will reveal the function of miR-130a in cancer, especially in drug resistance. Therefore, it will provide a new therapeutic target for the treatment of cancer, especially in chemotherapy.

Knockout of miR-221 and miR-222 reveals common and specific targets for paralogous miRNAs

ABSTRACTMicroRNAs (miRNAs) regulate the expression of mRNA through sequence-specific binding of the 3′ untranslated region (UTR). The seed sequence of miRNAs is the key determinant for target site recognition. Paralogous miRNAs, which share the same seed sequences but differ in their 3′ regions, are known to regulate largely overlapping groups of mRNAs. However, no study has analyzed functional differences between paralogous miRNAs with proper experimental methods. In this study, we compared the targets of paralogous miRNAs, miR-221 and miR-222. Using a nuclease-mediated genome engineering technique, we established knockout cell lines for these miRNAs, and precisely analyzed differences in target regulation. We found that miR-221 and miR-222 suppress the previously identified targets, CDKN1B and CDKN1C, differentially. Whereas both miRNAs suppressed CDKN1B, only miR-221 suppressed CDKN1C. From transcriptome analyses, we found that several different target mRNAs were regulated by each of miR-221 and miR-222 independently, although a large number of mRNAs responded commonly to miR-221 and miR-222. This is the first study to compare the mRNA regulations by paralogous miRNAs and illustrate that paralogous miRNAs with the same seed sequence also have difference in target regulation.

Cyclic Cationic Peptides Containing Sugar Amino Acids Selectively Distinguishes and Inhibits Maturation of Pre-miRNAs of the Same Family.

The discovery of microRNAs (miRNAs) has added a new dimension to the gene regulatory networks, making aberrantly expressed miRNAs as therapeutically important targets. Small molecules that can selectively target and modulate miRNA levels can thus serve as lead structures. Cationic cyclic peptides containing sugar amino acids represent a new class of small molecules that can target miRNA selectively. Upon treatment of these small molecules in breast cancer cell line, we profiled 96 therapeutically important miRNAs associated with cancer and observed that these peptides can selectively target paralogous miRNAs of the same seed family. This selective inhibition is of prime significance in cases when miRNAs of the same family have tissue-specific expression and perform different functions. During these conditions, targeting an entire miRNA family could lead to undesired adverse effects. The selective targeting is attributable to the difference in the three-dimensional structures of precursor miRNAs. Hence, the core structure of these peptides can be used as a scaffold for designing more potent inhibitors of miRNA maturation and hence function.

Discrimination of the Expression of Paralogous microRNA Precursors That Share the Same Major Mature Form

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs generated from endogenous transcripts that form hairpin structures. The hairpin precursor is processed into two mature miRNAs that form major/minor duplexes. Mature miRNAs regulate gene expression by cleaving mRNA or repressing protein translation. Numerous miRNAs have been discovered via deep sequencing. Many miRNAs are produced from multiple genome sites. These miRNAs are grouped into paralogous families of miRNAs that generate the same major mature form within organisms. Currently, no method of distinguishing the expression of these miRNAs is available.ResultsIn the present study, strategies were developed to discriminate and quantify the expression of paralogous miRNA precursors. First, paralogous miRNA precursors that were differentially expressed in tissues were identified through analysis of the coexpression scores of their major and minor forms based on deep sequencing data. Then the precursors were identified by monitoring the expression of their host gene or minor form using real-time PCR. Finally, precursors were identified by assessing the expression of clusters of miRNA members. These approaches were used to distinguish miR-128-1 and miR-128-2 as well as miR-194-1 and miR-194-2. The mechanism of transcription related to the differential expression of miR-194-1 and miR-194-2 was also investigated.ConclusionThis is the first report to distinguish paralogous miRNA copies by analyzing the expression of major-minor pairs, the host gene, and miRNA clusters. Discriminating paralogous precursors can provide useful information for investigating the mechanisms that regulate miRNA gene expression under different physiological and pathological conditions.

Close association between paralogous multiple isomiRs and paralogous/orthologues miRNA sequences implicates dominant sequence selection across various animal species

MicroRNAs (miRNAs) are crucial negative regulators of gene expression at the post-transcriptional level. Next-generation sequencing technologies have identified a series of miRNA variants (named isomiRs). In this study, paralogous isomiR assemblies (from the miRNA locus) were systematically analyzed based on data acquired from deep sequencing data sets. Evolutionary analysis of paralogous (members in miRNA gene family in a specific species) and orthologues (across different animal species) miRNAs was also performed. The sequence diversity of paralogous isomiRs was found to be similar to the diversity of paralogous and orthologues miRNAs. Additionally, both isomiRs and paralogous/orthologues miRNAs were implicated in 5′ and 3′ ends (especially 3′ ends), nucleotide substitutions, and insertions and deletions. Generally, multiple isomiRs can be produced from a single miRNA locus, but most of them had lower enrichment levels, and only several dominant isomiR sequences were detected. These dominant isomiR groups were always stable, and one of them would be selected as the most abundant miRNA sequence in specific animal species. Some isomiRs might be consistent to miRNA sequences in some species but not the other. Homologous miRNAs were often detected in similar isomiR repertoires, and showed similar expression patterns, while dominant isomiRs showed complex evolutionary patterns from miRNA sequences across the animal kingdom. These results indicate that the phenomenon of multiple isomiRs is not a random event, but rather the result of evolutionary pressures. The existence of multiple isomiRs enables different species to express advantageous sequences in different environments. Thus, dominant sequences emerge in response to functional and evolutionary pressures, allowing an organism to adapt to complex intra- and extra-cellular events.

Rapid intraspecific evolution of miRNA and siRNA genes in the mosquito Aedes aegypti.

RNA silencing, or RNA interference (RNAi) in metazoans mediates development, reduces viral infection and limits transposon mobility. RNA silencing involves 21–30 nucleotide RNAs classified into microRNA (miRNA), exogenous and endogenous small interfering RNAs (siRNA), and Piwi-interacting RNA (piRNA). Knock-out, silencing and mutagenesis of genes in the exogenous siRNA (exo-siRNA) regulatory network demonstrate the importance of this RNAi pathway in antiviral immunity in Drosophila and mosquitoes. In Drosophila, genes encoding components for processing exo-siRNAs are among the fastest evolving 3% of all genes, suggesting that infection with pathogenic RNA viruses may drive diversifying selection in their host. In contrast, paralogous miRNA pathway genes do not evolve more rapidly than the genome average. Silencing of exo-siRNA pathway genes in mosquitoes orally infected with arboviruses leads to increased viral replication, but little is known about the comparative patterns of molecular evolution among the exo-siRNA and miRNA pathways genes in mosquitoes. We generated nearly complete sequences of all exons of major miRNA and siRNA pathway genes dicer-1 and dicer-2, argonaute-1 and argonaute-2, and r3d1 and r2d2 in 104 Aedes aegypti mosquitoes collected from six distinct geographic populations and analyzed their genetic diversity. The ratio of replacement to silent amino acid substitutions was 1.4 fold higher in dicer-2 than in dicer-1, 27.4 fold higher in argonaute-2 than in argonaute-1 and similar in r2d2 and r3d1. Positive selection was supported in 32% of non-synonymous sites in dicer-1, in 47% of sites in dicer-2, in 30% of sites in argonaute-1, in all sites in argonaute-2, in 22% of sites in r3d1 and in 55% of sites in r2d2. Unlike Drosophila, in Ae. aegypti, both exo-siRNA and miRNA pathway genes appear to be undergoing rapid, positive, diversifying selection. Furthermore, refractoriness of mosquitoes to infection with dengue virus was significantly positively correlated for nucleotide diversity indices in dicer-2.

Abstract B21: The paralogous microRNA clusters, miR-17-92 and miR-106-25, are specifically overexpressed in metastatic non-small cell lung carcinomas

Abstract Background: Lung cancer is the cause of the most cancer-related deaths worldwide, with poor survival being largely attributed to late stage of disease at diagnosis and frequent metastasis. Understanding the mechanisms by which lung tumors metastasize could enable the development of anti-metastatic interventions or more specific therapeutics for metastatic disease. MicroRNAs (miRNAs) are major regulators of gene expression and control a wide range of cellular processes involved in metastasis, including apoptosis and cell cycle progression. Recently, increased expression of two paralogous miRNA clusters, miR-17-92 and miR-106b-25, was tied to control of these functions through antagonizing transforming growth factor-β (TGFβ) signaling. A tumor suppressive cytokine, TGFβ regulates cell cycle progression and apoptosis through activation of p21 and BIM, respectively. However, evidence suggests this regulation can be repressed by the overexpression of these microRNA clusters to promote tumor progression. To this end, we sought to determine whether or not expression of these clusters was increased in non-small cell lung cancer (NSCLC) cases positive for nodal or distant metastases compared to those with only locally invasive disease. Methods: A panel of 41 non-metastatic NSCLCs and a panel of 28 NSCLCs with nodal or distant metastases were collected along with paired adjacent non-malignant tissues. Expression analysis of miRNAs was conducted in these specimens using Illumina GaXII small RNA sequencing technologies. Matched tumor and normal miRNA normalized read count comparisons were performed for each miRNA in the miR-17-92 (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92a-1) and miR-106b-25 (miR-106b, miR-93, miR-25) clusters (Wilcoxon Signed-Rank test p<0.05). Only those miRNAs that were significantly overexpressed and displayed a minimum average expression fold change of 2 were further investigated. Results: The significant overexpression of several miRNAs occurred specifically in the metastatic cohort, and included miR-20a, miR-92a-1, miR-106b and miR-93. miR-18a and miR-19a were significantly overexpressed in both tumor cohorts as compared to matched normal tissue; however, expression levels were substantially higher in the metastatic cohort and increased by 9.5 and 3 fold, respectively. miR-17 and miR-19b-1 were upregulated in both cohorts to a similar level, while expression of miR-25 was not significantly altered. Conclusion: miRNAs in the miR-17-92 and miR-106b-25 clusters, save miR-25, were significantly overexpressed in NSCLC cases positive for nodal or distant metastases. There was an overall trend of increasing involvement of miRNAs from these clusters moving from NSCLC cases without metastases to those with metastases, suggesting upregulation of these miRNAs is involved in the metastatic process. Citation Format: Katey SS Enfield, Stephen Lam, Wan L. Lam. The paralogous microRNA clusters, miR-17-92 and miR-106-25, are specifically overexpressed in metastatic non-small cell lung carcinomas [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B21.

Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection.

MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes them virtually indistinguishable from one another by conventional hybridization-based approaches. Here we describe two inexpensive, sensitive methods for rapidly discriminating between paralogous miRNAs or other closely related miRNAs and for quantifying their abundance. The first approach is a sequential ribonuclease-protection and primer-extension assay; the second approach is a primer-extension assay that employs short oligonucleotide probes to exacerbate the instability of mismatched probe:miRNA hybrids. Both approaches are rapid and can be easily performed in their entirety using common laboratory equipment. As a proof of concept, we have used these methods to determine the exact identities of the human miR-17 family members that are increased by infection with the intracellular parasite Toxoplasma gondii. These methods can be used to rapidly and inexpensively discriminate between any closely related miRNAs in any organism.

Many Families of C. elegans MicroRNAs Are Not Essential for Development or Viability

MicroRNAs (miRNAs) are approximately 23 nt regulatory RNAs that posttranscriptionally inhibit the functions of protein-coding mRNAs. We previously found that most C. elegans miRNAs are individually not essential for development or viability and proposed that paralogous miRNAs might often function redundantly. To test this hypothesis, we generated mutant C. elegans strains that each lack multiple or all members of one of 15 miRNA families. Mutants for 12 of these families did not display strong synthetic abnormalities, suggesting that these miRNA families have subtle roles during development. By contrast, mutants deleted for all members of the mir-35 or mir-51 families died as embryos or early larvae, and mutants deleted for four members of the mir-58 family showed defects in locomotion, body size, and egg laying and an inability to form dauer larvae. Our findings indicate that the regulatory functions of most individual miRNAs and most individual families of miRNAs related in sequence are not critical for development or viability. Conversely, because in some cases miRNA family members act redundantly, our findings emphasize the importance of determining miRNA function in the absence of miRNAs related in sequence.

MicroRNA (miRNA) Transcriptome of Mouse Retina and Identification of a Sensory Organ-specific miRNA Cluster

Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6).