Canonical microRNA Biogenesis Research Articles

TRNA, yRNA, and rRNA fragment excisions do not involve canonical microRNA biogenesis machinery.

The excision of specific tRNA-derived small RNAs (tsRNAs), yRNA-derived small RNAs (ysRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) is now well established. Several reports have suggested many of these fragments function much like traditional microRNAs (miRNAs). That said, whereas the expressions of the majority of appreciably expressed miRNAs in HCT116 colon cancer cells are significantly decreased in individual knockouts (KOs) of DROSHA, DGCR8, XPO5, and DICER, on average, only 3.5% of tsRNA, ysRNA, and rsRNA expressions are impaired. Conversely, tsRNA, ysRNA, and rsRNA expressions are significantly increased in each of these KOs as compared to WT. As such, although DICER has been suggested to be involved with the expression of specific tsRNAs, ysRNAs, and rsRNAs, our study finds no evidence supporting the involvement of any of these canonical miRNA biogenesis enzymes in their expressions.

MicroRNA Biogenesis in Cell Senescence Induced by Chronic Endoplasmic Reticulum Stress

MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal β-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice.

ObjectivesThe female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract.Materials and Methods Dgcr8 f/f mice were crossed with Ltf icre/+, Amhr2 cre/+ or PR cre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8 ed/ed), mesenchymal (Dgcr8 md/md) and all the compartments (Dgcr8 td/td) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA‐seq, mRNA‐seq, real‐time RT‐PCR, and/or morphologic and histological analyses.Result Dgcr8 ed/ed mice did not exhibit any distinct defects, whereas Dgcr8 md/md mice showed sub‐fertility and oviductal smooth muscle deformities. Dgcr8 td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8 td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8 td/td uterus were different from those in other genotypes in a Cre‐dependent manner.Conclusions Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.

MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi

BackgroundMicroRNAs play a vital role in the regulation of gene expression and have been identified in every animal with a sequenced genome examined thus far, except for the placozoan Trichoplax. The genomic repertoires of metazoan microRNAs have become increasingly endorsed as phylogenetic characters and drivers of biological complexity.ResultsIn this study, we report the first investigation of microRNAs in a species from the phylum Ctenophora. We use short RNA sequencing and the assembled genome of the lobate ctenophore Mnemiopsis leidyi to show that this species appears to lack any recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. This finding represents the first reported case of a metazoan lacking a Drosha protein.ConclusionsRecent phylogenomic analyses suggest that Mnemiopsis may be the earliest branching metazoan lineage. If this is true, then the origins of canonical microRNA biogenesis and microRNA-mediated gene regulation may postdate the last common metazoan ancestor. Alternatively, canonical microRNA functionality may have been lost independently in the lineages leading to both Mnemiopsis and the placozoan Trichoplax, suggesting that microRNA functionality was not critical until much later in metazoan evolution.

Biogenesis of mammalian microRNAs by a non-canonical processing pathway

Canonical microRNA biogenesis requires the Microprocessor components, Drosha and DGCR8, to generate precursor-miRNA, and Dicer to form mature miRNA. The Microprocessor is not required for processing of some miRNAs, including mirtrons, in which spliceosome-excised introns are direct Dicer substrates. In this study, we examine the processing of putative human mirtrons and demonstrate that although some are splicing-dependent, as expected, the predicted mirtrons, miR-1225 and miR-1228, are produced in the absence of splicing. Remarkably, knockout cell lines and knockdown experiments demonstrated that biogenesis of these splicing-independent mirtron-like miRNAs, termed ‘simtrons’, does not require the canonical miRNA biogenesis components, DGCR8, Dicer, Exportin-5 or Argonaute 2. However, simtron biogenesis was reduced by expression of a dominant negative form of Drosha. Simtrons are bound by Drosha and processed in vitro in a Drosha-dependent manner. Both simtrons and mirtrons function in silencing of target transcripts and are found in the RISC complex as demonstrated by their interaction with Argonaute proteins. These findings reveal a non-canonical miRNA biogenesis pathway that can produce functional regulatory RNAs.