Characterization of the mammalian miRNA turnover landscape

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5′ nucleotide bias against Argonaute-(AGO)-loading, but also additional 3′ and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.     

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the “RIP-Chip” assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.     

Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3′-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.     

Analysis of microRNA turnover in mammalian cells following Dicer1 ablation

Although microRNAs (miRNAs) are key regulators of gene expression, little is known of their overall persistence in the cell following processing. Characterization of such persistence is key to the full appreciation of their regulatory roles. Accordingly, we measured miRNA decay rates in mouse embryonic fibroblasts following loss of Dicer1 enzymatic activity. The results confirm the inherent stability of miRNAs, the intracellular levels of which were mostly affected by cell division. Using the decay rates of a panel of six miRNAs representative of the global trend of miRNA decay, we establish a mathematical model of miRNA turnover and determine an average miRNA half-life of 119 h (i.e. ∼5 days). In addition, we demonstrate that select miRNAs turnover more rapidly than others. This study constitutes, to our knowledge, the first in-depth characterization of miRNA decay in mammalian cells. Our findings indicate that miRNAs are up to 10× more stable than messenger RNA and support the existence of novel mechanism(s) controlling selective miRNA cellular concentration and function.     

Systematic Analysis of Small RNAs Associated with Human Mitochondria by Deep Sequencing: Detailed Analysis of Mitochondrial Associated miRNA

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.     

Precursor microRNA-programmed silencing complex assembly pathways in mammals

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in?vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in?vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to?miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes?miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.     

Numerous microRNPs in neuronal cells containing novel microRNAs

Spinal muscular atrophy (SMA) is a common neurodegenerative disease that is caused by deletions or loss-of-function mutations in the Survival of Motor Neuron (SMN) protein. SMN is part of a large complex that functions in the assembly/restructuring of ribonucleoprotein (RNP) complexes. We recently showed in HeLa cells that two components of the SMN complex, Gemin3 and Gemin4, together with the argonaute protein eIF2C2, also associate with microRNAs (miRNAs) as part of a novel class of RNPs termed miRNPs. Here we report on miRNPs isolated from neuronal cell lines of mouse and human, and describe 53 novel miRNAs. Several of these miRNAs are conserved in divergent organisms, including rat, zebrafish, pufferfish, and the nematode Caenorhabditis elegans. The chromosomal locations of most of the novel miRNAs were identified and indicate some phylogenetic conservation of the likely precursor structures. Interestingly the gene locus of one miRNA, miR-175, is a candidate region for two neurologic diseases: early-onset parkinsonism (Waisman syndrome) and X-linked mental retardation (MRX3). Also, several miRNAs identified as part of miRNPs in these cells appear to constitute two distinct subfamilies. These subfamilies comprise multiple copies of miRNAs on different chromosomes, suggesting an important function in the regulation of gene expression.     

Site-specific crosslinking of human microRNPs to RNA targets

MicroRNAs (miRNAs) regulate the expression of numerous genes and are implicated in the pathogenesis of many human diseases. miRNAs act as specificity determinants to guide deposition of microRNPs (miRNPs) onto miRNA recognition elements (MREs) that are found in mRNA targets. We have adapted a site-specific crosslinking approach, previously used in the analysis of splicing, to interrogate protein factors that physically associate with MREs in human cells. We find that Ago proteins are the only proteins that bind to the MRE in a miRNA-dependent manner. Our method may be used in various experimental systems to analyze protein factors that influence MRE accessibility by miRNAs and the composition of MRE-bound miRNPs.     

Impact of miRNA Sequence on miRNA Expression and Correlation between miRNA Expression and Cell Cycle Regulation in Breast Cancer Cells

The miRNAs regulate cell functions by inhibiting expression of proteins. Research on miRNAs had usually focused on identifying targets by base pairing between miRNAs and their targets. Instead of identifying targets, this paper proposed an innovative approach, namely impact significance analysis, to study the correlation between mature sequence, expression across patient samples or time and global function on cell cycle signaling of miRNAs. With three distinct types of data: The Cancer Genome Atlas miRNA expression data for 354 human breast cancer specimens, microarray of 266 miRNAs in mouse Embryonic Stem cells (ESCs), and Reverse Phase Protein Array (RPPA) transfected by 776 miRNAs in MDA-MB-231 cell line, we linked the expression and function of miRNAs by their mature sequence and discovered systematically that the similarity of miRNA expression enhances the similarity of miRNA function, which indicates the miRNA expression can be used as a supplementary factor to predict miRNA function. The results also show that both seed region and 3'' portion are associated with miRNA expression levels across human breast cancer specimens and in ESCs; miRNAs with similar seed tend to have similar 3'' portion. And we discussed that the impact of 3'' portion, including nucleotides , is not significant for miRNA function. These results provide novel insights to understand the correlation between miRNA sequence, expression and function. They can be applied to improve the prediction algorithm and the impact significance analysis can also be implemented to similar analysis for other small RNAs such as siRNAs.     

Real-Time Monitoring of miRNA Function in Pancreatic Cell Lines Using Recombinant AAV-Based miRNA Asensors

Background

MicroRNAs (miRNAs) are reportedly involved in pancreatic ductal adenocarcinoma (PDAC) development. Current methods do not allow us to reliably monitor miRNA function. Asensors are adeno-associated virus (AAV) vector miRNA sensors for real-time consecutive functional monitoring of miRNA profiling in living cells.

Methods

miR-200a, -200b, -21, -96, -146a, -10a, -155, and -221 in three PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma cells (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were monitored by Asensors. Subsequently, the real-time consecutive functional profile of all miRNAs was evaluated.

Results

Selected miRNAs were detectable in all cell lines with high sensitivity and reproducibility. In the three PDAC cell lines, BxPC-3, CFPAC-1, and SW1990, the calibrated signal unit of the eight miRNAs Asensors was significantly lower than that of the Asensor control. However, in PANC-1 cells, miR-200a and -155 showed upregulation of target gene expression at 24 hours after infection with the sensors; at 48 hours, miR-200b and -155 displayed upregulation of reporter expression; and at 72 hours, reporter gene expression was upregulated by miR-200a and -200b. The result that miRNA could upregulate gene expression was further confirmed in miR-155 of hTERT-HPNE cells. Furthermore, miRNA activity varied among cell/tissue types and time.

Conclusion

It is possible that miRNA participates in the pathophysiology of pancreatic cancer, but the current popular methods do not accurately reveal the real-time miRNA function. Thus, this report provided a convenient, accurate, and sensitive approach to miRNA research.     

The role of miRNA in the direct and indirect effects of ionizing radiation

This review focuses on a number of recent studies that have examined changes in microRNA (miRNA) expression profiles in response to ionizing radiation and other forms of oxidative stress. In both murine and human cells and tissues, a number of miRNAs display significant alterations in expression levels in response to both direct and indirect radiation exposure. In terms of direct irradiation, or exposure to agents that induce oxidative stress, miRNA array analyses indicate that a number of miRNAs are up- and down-regulated and, in particular, the let-7 family of miRNAs may well be critical in the cellular response to oxidative stress. In bystander cells that are not directly irradiated, but close to, or share media with directly irradiated cells or tissues, the miRNA expression profiles are also altered, but are somewhat distinct from the directly irradiated cells. Based on the results of these numerous studies, as well as our own data presented here, we conclude that miRNA regulation is a critical step in the cellular response to radiation and oxidative stress and that future studies should elucidate the mechanisms through which this altered regulation affects cell metabolism.     

miRNA sensitivity to Drosha levels correlates with pre-miRNA secondary structure

microRNAs (miRNAs) are crucial for cellular development and homeostasis. In order to better understand regulation of miRNA biosynthesis, we studied cleavage of primary miRNAs by Drosha. While Drosha knockdown triggers an expected decrease of many mature miRNAs in human embryonic stem cells (hESC), a subset of miRNAs are not reduced. Statistical analysis of miRNA secondary structure and fold change of expression in response to Drosha knockdown showed that absence of mismatches in the central region of the hairpin, 5 and 9–12 nt from the Drosha cutting site conferred decreased sensitivity to Drosha knockdown. This suggests that, when limiting, Drosha processes miRNAs without mismatches more efficiently than mismatched miRNAs. This is important because Drosha expression changes over cellular development and the fold change of expression for miRNAs with mismatches in the central region correlates with Drosha levels. To examine the biochemical relationship directly, we overexpressed structural variants of miRNA-145, miRNA-137, miRNA-9, and miRNA-200b in HeLa cells with and without Drosha knockdown; for these miRNAs, elimination of mismatches in the central region increased, and addition of mismatches decreased their expression in an in vitro assay and in cells with low Drosha expression. Change in Drosha expression can be a biologically relevant mechanism by which eukaryotic cells control miRNA profiles. This phenomenon may explain the impact of point mutations outside the seed region of certain miRNAs.     

The miRNA as human cell gene activity regulator after ionizing radiation

This review presents analysis of the literature and our own research with respect to the role of miRNAs in the regulation of activity (expression) of genes controlling cellular homeostasis in human cells when exposed to ionizing radiation. Human cells, on one hand, can have increased resistance to radiation, which hinders the effectiveness of tumor treatment in radiotherapy. On the other hand, increased sensitivity to radiation may be accompanied by the development of several pathologies, including tumorigenesis. This paper examines the role of specific miRNAs in the formation of radioresistance and radiosensitivity of human cells and their impact on the respective target genes. Separate sections are devoted to the role of different miRNAs in radiation therapy of tumors of different localization, as well as their role in the bystander effect. A special section highlights features of gene activity and its regulators, miRNAs, in radiosensitive cells in patients with Down syndrome. The final section provides information about new approaches to change miRNA expression and, accordingly, their target genes by the action of plant and synthetic drugs (crown compounds) which reduce damaging effects of mutagens. It is assumed that antimutagens affecting the expression levels of miRNAs and structural genes may be used to correct the increase and decrease in cellular radioresponse, reducing the risk of development of pathological processes, including tumorigenesis.