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Bulletin of Experimental Biology and... May 2022We studied the correlations between the levels of methylation of a group of 21 microRNA genes in 99 primary tumors and 29 macroscopic peritoneal metastases of ovarian...
We studied the correlations between the levels of methylation of a group of 21 microRNA genes in 99 primary tumors and 29 macroscopic peritoneal metastases of ovarian cancer. Analysis of the level of methylation by quantitative methylation-specific PCR showed that co-methylation was detected for 13 pairs of microRNA genes in primary tumors and for 22 pairs in metastases. Pairs of microRNA genes that have shown significant co-methylation can be involved in common processes and pathways of gene regulation and interaction and can have common target genes. The results are highly significant and pairs of microRNA genes can be proposed as new potential markers for the diagnosis and prognosis of ovarian cancer metastasis.
Topics: Carcinoma, Ovarian Epithelial; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Ovarian Neoplasms
PubMed: 35622253
DOI: 10.1007/s10517-022-05499-y -
Cells Jan 2023MicroRNAs (miRNAs) are versatile, post-transcriptional regulators of gene expression. Canonical miRNAs are generated through the two-step DROSHA- and DICER-mediated... (Review)
Review
MicroRNAs (miRNAs) are versatile, post-transcriptional regulators of gene expression. Canonical miRNAs are generated through the two-step DROSHA- and DICER-mediated processing of primary miRNA (pri-miRNA) transcripts with optimal or suboptimal features for DROSHA and DICER cleavage and loading into Argonaute (AGO) proteins, whereas multiple hairpin-structured RNAs are encoded in the genome and could be a source of non-canonical miRNAs. Recent advances in miRNA biogenesis research have revealed details of the structural basis of miRNA processing and cluster assistance mechanisms that facilitate the processing of suboptimal hairpins encoded together with optimal hairpins in polycistronic pri-miRNAs. In addition, a deeper investigation of miRNA-target interaction has provided insights into the complexity of target recognition with distinct outcomes, including target-mediated miRNA degradation (TDMD) and cooperation in target regulation by multiple miRNAs. Therefore, the coordinated or network regulation of both miRNA biogenesis and miRNA-target interaction is prevalent in miRNA biology. Alongside recent advances in the mechanistic investigation of miRNA functions, this review summarizes recent findings regarding the ordered regulation of miRNA biogenesis and miRNA-target interaction.
Topics: MicroRNAs; RNA Processing, Post-Transcriptional
PubMed: 36672241
DOI: 10.3390/cells12020306 -
Nature Communications Jan 2022Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human...
Abnormalities of ventricular action potential cause malignant cardiac arrhythmias and sudden cardiac death. Here, we aim to identify microRNAs that regulate the human cardiac action potential and ask whether their manipulation allows for therapeutic modulation of action potential abnormalities. Quantitative analysis of the microRNA targetomes in human cardiac myocytes identifies miR-365 as a primary microRNA to regulate repolarizing ion channels. Action potential recordings in patient-specific induced pluripotent stem cell-derived cardiac myocytes show that elevation of miR-365 significantly prolongs action potential duration in myocytes derived from a Short-QT syndrome patient, whereas specific inhibition of miR-365 normalizes pathologically prolonged action potential in Long-QT syndrome myocytes. Transcriptome analyses in these cells at bulk and single-cell level corroborate the key cardiac repolarizing channels as direct targets of miR-365, together with functionally synergistic regulation of additional action potential-regulating genes by this microRNA. Whole-cell patch-clamp experiments confirm miR-365-dependent regulation of repolarizing ionic current I. Finally, refractory period measurements in human myocardial slices substantiate the regulatory effect of miR-365 on action potential in adult human myocardial tissue. Our results delineate miR-365 to regulate human cardiac action potential duration by targeting key factors of cardiac repolarization.
Topics: Action Potentials; Arrhythmias, Cardiac; Gene Expression Profiling; HEK293 Cells; Heart Ventricles; Humans; Long QT Syndrome; MicroRNAs; Myocardium; Myocytes, Cardiac
PubMed: 35017523
DOI: 10.1038/s41467-021-27856-7 -
Nature Structural & Molecular Biology Sep 2008microRNAs (miRNAs) are generated from long primary (pri-) RNA polymerase II (Pol II)-derived transcripts by two RNase III processing reactions: Drosha cleavage of...
microRNAs (miRNAs) are generated from long primary (pri-) RNA polymerase II (Pol II)-derived transcripts by two RNase III processing reactions: Drosha cleavage of nuclear pri-miRNAs and Dicer cleavage of cytoplasmic pre-miRNAs. Here we show that Drosha cleavage occurs during transcription acting on both independently transcribed and intron-encoded miRNAs. We also show that both 5'-3' and 3'-5' exonucleases associate with the sites where co-transcriptional Drosha cleavage occurs, promoting intron degradation before splicing. We finally demonstrate that miRNAs can also derive from 3' flanking transcripts of Pol II genes. Our results demonstrate that multiple miRNA-containing transcripts are co-transcriptionally cleaved during their synthesis and suggest that exonucleolytic degradation from Drosha cleavage sites in pre-mRNAs may influence the splicing and maturation of numerous mRNAs.
Topics: Chromatin; Chromatin Immunoprecipitation; HeLa Cells; Humans; Introns; MicroRNAs; Microtubule-Associated Proteins; Nucleic Acid Conformation; RNA Polymerase II; RNA Splicing; Ribonuclease III; Transcription, Genetic; beta-Globins
PubMed: 19172742
DOI: 10.1038/nsmb.1475 -
STAR Protocols Mar 2022We describe a protocol to conduct a high-throughput processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed...
We describe a protocol to conduct a high-throughput processing assay, using 1,881 human primary microRNAs (pri-miRNAs) and recombinant Microprocessor complex, followed by deep sequencing library generation. This comprehensive approach allows the mapping of cleavage sites and the measurement of processing efficiency of a large number of substrates simultaneously. Our protocol is readily modifiable to investigate the effects of chemicals and regulatory proteins. Moreover, -acting elements can be examined by replacing the wild-type pri-miRNAs with mutant variants. For complete details on the use and execution of this profile, please refer to Kim et al. (2021).
Topics: Humans; MicroRNAs; Microcomputers; Ribonuclease III
PubMed: 35036952
DOI: 10.1016/j.xpro.2021.101042 -
Scientific Reports Jul 2015Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur...
Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.
Topics: HeLa Cells; Humans; MicroRNAs; Nucleic Acid Conformation; RNA Polymerase II; RNA Precursors; RNA Processing, Post-Transcriptional; Transcription, Genetic
PubMed: 26149087
DOI: 10.1038/srep11992 -
Methods in Molecular Biology (Clifton,... 2006MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter... (Review)
Review
MicroRNAs (miRNAs) are approx 22-nucleotide (nt)-long, single-stranded, endogenous, noncoding RNAs that are widely expressed in multicellular organisms. This chapter describes methods that allow the overexpression of human miRNAs and also discusses how primary miRNAs (pri-miRNAs), the much longer precursors of mature miRNAs, are processed in human cells, as well as in vitro.
Topics: Humans; In Vitro Techniques; MicroRNAs; RNA Interference; RNA Precursors; RNA Processing, Post-Transcriptional; Ribonuclease III
PubMed: 16957366
DOI: 10.1385/1-59745-123-1:49 -
Journal of Genetics and Genomics = Yi... Feb 2023Single-nucleus RNA-sequencing technology has revolutionized understanding of nuanced changes in gene expression between cell types within tissues. Unfortunately, our...
Single-nucleus RNA-sequencing technology has revolutionized understanding of nuanced changes in gene expression between cell types within tissues. Unfortunately, our understanding of regulatory RNAs, such as microRNAs (miRNAs), is limited through both single-cell and single-nucleus techniques due to the short length of miRNAs in the cytoplasm and the incomplete reference of longer primary miRNA (pri-miRNA) transcripts in the nucleus. We build a custom reference to align and count pri-miRNA sequences in single-nucleus data. Using young and aged subventricular zone (SVZ) nuclei, we show differential expression of pri-miRNAs targeting genes involved in neural stem cells (NSC) differentiation in the aged SVZ. Furthermore, using wild-type and 5XFAD mouse model cortex nuclei, to validate the use of primiReference, we find cell-type-specific expression of pri-miRNAs known to be involved in Alzheimer's disease (AD). pri-miRNAs likely contribute to NSC dysregulation with age and AD pathology. primiReference is paramount in capturing a global profile of gene expression and regulation in single-nucleus data and can provide key insights into cell-type-specific expression of pri-miRNAs, paving the way for future studies of regulation and pathway dysregulation. By looking at pri-miRNA abundance and transcriptional differences, regulation of gene expression by miRNAs in disease and aging can be further explored.
Topics: Animals; Mice; MicroRNAs; Sequence Analysis, RNA
PubMed: 36371075
DOI: 10.1016/j.jgg.2022.10.003 -
Biochemical Society Transactions Aug 2013The let-7 miRNA (microRNA) is an essential regulator of development from nematode worms to humans. Altered expression of let-7 results in larval arrest or lethality in...
The let-7 miRNA (microRNA) is an essential regulator of development from nematode worms to humans. Altered expression of let-7 results in larval arrest or lethality in Caenorhabditis elegans. Likewise, under- or over-expression of let-7 in human cells can result in cellular overproliferation or halted cell division respectively. Thus the biogenesis of this critical miRNA is controlled at multiple levels. An unexpected mechanism for regulating the initial processing of let-7 was recently found to involve the let-7 miRNA itself. The mature let-7 miRNA along with its effector protein, Argonaute, were shown to bind to a site in the primary transcripts produced by the let-7 gene. This interaction enhances processing through a novel auto-regulatory feedback loop. This discovery highlights a new role for the miRNA complex in regulating miRNA biogenesis and enriches the classes of RNAs targeted by Argonaute.
Topics: Animals; Caenorhabditis elegans; Humans; MicroRNAs
PubMed: 23863138
DOI: 10.1042/BST20130020 -
EBioMedicine Jun 2022Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic...
BACKGROUND
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs).
METHODS
Our study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment.
FINDINGS
Mature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5' and 3' strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47.
INTERPRETATION
We found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS.
FUNDING
This study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern.
Topics: Binding Sites; Gene Expression Profiling; Humans; MicroRNAs; Multiple Sclerosis; Polymorphism, Single Nucleotide
PubMed: 35561450
DOI: 10.1016/j.ebiom.2022.104052