Mapping and expression studies of the mir17-92 cluster on pig Chromosome 11

We have identified the first porcine microRNA (miRNA) cluster (the mir17-92 cluster) and localized it to the q-arm of pig Chromosome 11. The miRNA cluster was found by sequence similarity search with human miRNA sequences against the pig genomic data generated within the Sino-Danish pig genome project. The resulting data contained three complete and two incomplete miRNA precursors of seven miRNAs from the human mir17-92 cluster. Because there is a 100% sequence identity between the four pig miRNAs and the corresponding human miRNAs, the sequences of three unavailable pig miRNAs were derived from the human data. The expression profiles of seven studied miRNAs were analyzed by hybridization to Northern blots containing five porcine tissues: cerebellum, cortex, hippocampus, kidney, and liver. In order to determine the localization of the mir17-92 cluster in the pig genome, we mapped it by PCR in the porcine somatic cell hybrid (SCH) panel and in the INRA-University of Minnesota (INRA-UMN) porcine radiation hybrid (IMpRH) panel. The PCR results enabled us to localize this cluster to the q-arm of pig Chromosome 11 and map it in relation to two microsatellites. Our study presents the first expression analyses of miRNAs in pig and adds information for further functional studies in this species.     

Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication

The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.     

Human T-cell leukemia virus type I infects human lung epithelial cells and induces gene expression of cytokines, chemokines and cell adhesion molecules

Background

Cellular miRNAs play an important role in the regulation of gene expression in eukaryotes. Recently, miRNAs have also been shown to be able to target and inhibit viral gene expression. Computational predictions revealed earlier that the HIV-1 genome includes regions that may be potentially targeted by human miRNAs. Here we report the functionality of predicted miR-29a target site in the HIV-1 nef gene.

Results

We find that the human miRNAs hsa-miR-29a and 29b are expressed in human peripheral blood mononuclear cells. Expression of a luciferase reporter bearing the nef miR-29a target site was decreased compared to the luciferase construct without the target site. Locked nucleic acid modified anti-miRNAs targeted against hsa-miR-29a and 29b specifically reversed the inhibitory effect mediated by cellular miRNAs on the target site. Ectopic expression of the miRNA results in repression of the target Nef protein and reduction of virus levels.

Conclusion

Our results show that the cellular miRNA hsa-miR29a downregulates the expression of Nef protein and interferes with HIV-1 replication.     

Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.     

TDP-43 regulates cancer-associated microRNAs

Aberrant regulation of miRNA genes contributes to pathogenesis of a wide range of human diseases, including cancer. The TAR DNA binding protein 43 (TDP-43), a RNA/DNA binding protein associated with neurodegeneration, is involved in miRNA biogenesis. Here, we systematically examined miRNAs regulated by TDP-43 using RNA-Seq coupled with an siRNA-mediated knockdown approach. TDP-43 knockdown affected the expression of a number of miRNAs. In addition, TDP-43 down-regulation led to alterations in the patterns of different isoforms of miRNAs (isomiRs) and miRNA arm selection, suggesting a previously unknown role of TDP-43 in miRNA processing. A number of TDP-43 associated miRNAs, and their candidate target genes, are associated with human cancers. Our data reveal highly complex roles of TDP-43 in regulating different miRNAs and their target genes. Our results suggest that TDP-43 may promote migration of lung cancer cells by regulating miR-423-3p. In contrast, TDP-43 increases miR-500a-3p expression and binds to the mature miR-500a-3p sequence. Reduced expression of miR-500a-3p is associated with poor survival of lung cancer patients, suggesting that TDP-43 may have a suppressive role in cancer by regulating miR-500a-3p. Cancer-associated genes LIF and PAPPA are possible targets of miR-500a-3p. Our work suggests that TDP-43-regulated miRNAs may play multifaceted roles in the pathogenesis of cancer.     

Clustering and conservation patterns of human microRNAs

MicroRNAs (miRNAs) are ~22 nt-long non-coding RNA molecules, believed to play important roles in gene regulation. We present a comprehensive analysis of the conservation and clustering patterns of known miRNAs in human. We show that human miRNA gene clustering is significantly higher than expected at random. A total of 37% of the known human miRNA genes analyzed in this study appear in clusters of two or more with pairwise chromosomal distances of at most 3000 nt. Comparison of the miRNA sequences with their homologs in four other organisms reveals a typical conservation pattern, persistent throughout the clusters. Furthermore, we show enrichment in the typical conservation patterns and other miRNA-like properties in the vicinity of known miRNA genes, compared with random genomic regions. This may imply that additional, yet unknown, miRNAs reside in these regions, consistent with the current recognition that there are overlooked miRNAs. Indeed, by comparing our predictions with cloning results and with identified miRNA genes in other mammals, we corroborate the predictions of 18 additional human miRNA genes in the vicinity of the previously known ones. Our study raises the proportion of clustered human miRNAs that are <3000 nt apart to 42%. This suggests that the clustering of miRNA genes is higher than currently acknowledged, alluding to its evolutionary and functional implications.