3' Uridylation Confers miRNAs with Non-canonical Target Repertoires.

Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3' uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.

Flexible pri-miRNA structures enable tunable production of 5' isomiRs.

The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5' isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5' isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5' isomiR biogenesis in tumours.[Figure: see text].

Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.

Cytoplasmic Drosha activity generated by alternative splicing.

RNase III enzyme Drosha interacts with DGCR8 to form the Microprocessor, initiating canonical microRNA (miRNA) maturation in the nucleus. Here, we re-evaluated where Drosha functions in cells using Drosha and/or DGCR8 knock out (KO) cells and cleavage reporters. Interestingly, a truncated Drosha mutant located exclusively in the cytoplasm cleaved pri-miRNA effectively in a DGCR8-dependent manner. In addition, we demonstrated that in vitro generated pri-miRNAs when transfected into cells could be processed to mature miRNAs in the cytoplasm. These results indicate the existence of cytoplasmic Drosha (c-Drosha) activity. Although a subset of endogenous pri-miRNAs become enriched in the cytoplasm of Drosha KO cells, it remains unclear whether pri-miRNA processing is the main function of c-Drosha. We identified two novel in-frame Drosha isoforms generated by alternative splicing in both HEK293T and HeLa cells. One isoform loses the putative nuclear localization signal, generating c-Drosha. Further analysis indicated that the c-Drosha isoform is abundant in multiple cell lines, dramatically variable among different human tissues and upregulated in multiple tumors, suggesting that c-Drosha plays a unique role in gene regulation. Our results reveal a new layer of regulation on the miRNA pathway and provide novel insights into the ever-evolving functions of Drosha.

RNA⁻Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics.

Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of RNA fragments longer than 18 nucleotides that were not degraded by single-strand cutting nucleases. We applied this approach to four different cell lines with respect to three different treatments (native cell lysate, removal of proteins, and removal of ribosomal RNA and proteins). We found that long accessible RNA duplexes were largely absent in native cell lysates, while the number of RNA duplexes was dramatically higher when proteins were removed. The majority of RNA duplexes involved ribosomal transcripts. The duplex formation between different non-ribosomal transcripts appears to be largely of a stochastic nature. These results suggest that cells are-via RNA-binding proteins-mostly devoid of long RNA duplexes, leading to low "noise" in the molecular patterns that are utilized by the innate immune system. These findings have implications for the design of RNA interference (RNAi)-based therapeutics by imposing structural constraints on designed RNA complexes that are intended to have specific properties with respect to Dicer cleavage and target gene downregulation.

Regulatory Role of miRNA-375 in Expression of BMP15/GDF9 Receptors and its Effect on Proliferation and Apoptosis of Bovine Cumulus Cells.

BACKGROUND: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are members of the transforming growth factor beta (TGF-ß) superfamily. Through autocrine and paracrine mechanisms, these two factors can regulate cell differentiation, proliferation, and other functions in the ovary locally. Furthermore, GDF9 and BMP15 play vital roles in follicular growth, atresia, ovulation, fertilization, reproduction, and maintenance. Numerous studies have demonstrated a synergy between BMP15 and GDF9. Studies in humans and mice have indicated that the synergy between BMP15 and GDF9 is primarily mediated by the bone morphogenetic protein type II receptor (BMPR2). The BMP15/GDF9 heterodimer needs to bind to the BMPR2-ALK4/5/7-ALK6 receptor complex to activate the SMAD2/3 signaling pathway. However, it is not clear which genes mediate and regulate the effects of the BMP15/GDF9 proteins on bovine cumulus cells (CCs). METHODS: Our earlier study showed that BMPR2 is a gene that is directly targeted and regulated by miR-375. Therefore, we designed and synthesized an miR-375 mimics/inhibitor and regulated BMPR2 expression in bovine CCs by the overexpression or inhibition of miR-375. After the overexpression or inhibition of miR-375, the apoptosis rate of bovine CCs was measured by flow cytometry; changes in critical gene expression were measured by RT-qPCR and western blot assays; and the proliferation of bovine CCs was measured by CCK-8 assay. RESULTS: In bovine CCs, the overexpression of miR-375 resulted in decreased BMPR2 and ALK7 expression, whereas the inhibition of miR-375 caused increased BMPR2 and ALK7 expression. The overexpression of miR-375 attenuated the proliferation ability and significantly increased the apoptosis rate of bovine CCs, whereas the inhibition of miR-375 did not significantly change the proliferation ability or apoptosis rate. CONCLUSIONS: BMPR2, a target of miR-375, is regulated by this molecule, thereby affecting expression of BMP15/GDF9 receptors, and the proliferation and apoptosis of bovine CCs.

The possible FAT1-mediated apoptotic pathways in porcine cumulus cells.

Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell contact, actin dynamics, and cell growth suppression. To understand whether the FAT1 gene affects the apoptosis of porcine cumulus cells and to elucidate the mechanism of this potential action, FAT1 was knocked down using RNA interference. The lack of FAT1 resulted in stable expression of CTNNB, enhanced expression of cleaved CASP3, but decreased the BCL2/BAX ratios at both the mRNA and protein levels. These results indicated that FAT1 inhibited porcine cumulus cell apoptosis via different pathways. Taken together, these data provide new insights into the mechanisms of the association between FAT1 and porcine cumulus cell apoptosis.

Thymine DNA Glycosylase Gene Knockdown Can Affect the Differentiation of Pig Preadipocytes.

AIMS: To study the effect of thymine DNA glycosylase (TDG) gene knockdown on the differentiation of pig preadipocytes. METHODS: Preadipocytes were obtained from subcutaneous adipose tissue from the neck of 1- to 7-day-old pigs. The TDG gene was knocked down using siRNA, and cell differentiation was induced. The mRNA expression level was measured using fluorescence quantitative PCR, and the protein expression level was determined using Western blot analysis. The DNA methylation levels in promoter regions of differentiation-related genes were also evaluated. RESULTS: TDG gene knockdown decreased the mRNA expression levels of the peroxisome proliferator-activated receptorγ (PPARγ) and Fatty acid binding proteins 4(FABP4 Also known as aP2) genes (P<0.01), while the mRNA expression level of the CCAAT/enhancer binding protein alpha(C/EBPα) gene did not change significantly (P>0.05). In addition, after induced differentiation, the lipid droplet production significantly decreased, and the percentages of methylation in the promoter regions of C/EBPα, PPARγ, and aP2 genes were 0.9%, 80%, and 76%, respectively. In contrast, the percentages of methylation in the negative control groups were 0.5%, 67.5%, and 58%, respectively. CONCLUSION: TDG gene knockdown could inhibit the differentiation of pig preadipocytes and affect the DNA methylation levels of some transcription factors.

MiR-378 Plays an Important Role in the Differentiation of Bovine Preadipocytes.

BACKGROUND: Adipocyte, the main cellular component of white adipose tissue, plays a vital role in energy balance in higher eukaryotes. In recent years, adipocytes have also been identified as a major endocrine organ involved in immunological responses, vascular diseases, and appetite regulation. In farm animals, fat content and categories are closely correlated with meat quality. MicroRNAs (miRNAs), a class of endogenous single-stranded non-coding RNA molecules, participate in the regulation of adipocyte differentiation and adipogenesis through regulating the transcription or translation of target mRNAs. MiR-378 plays an important role in a number of biological processes, including cell growth, cell differentiation, tumor cell survival and angiogenesis. METHODS: In the present study, bioinformatics analysis and dual-luciferase reporter assay were used to identify and validate the target genes of miR-378. In vitro cell transfection, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis, Oil Red O staining, and triglyceride content measurement were conducted to analyze the effects of miR-378 on bovine preadipocyte differentiation. RESULTS: MiR-378 was induced during adipocyte differentiation. In the differentiated adipocytes overexpressing miR- 378, the volume of lipid droplets was enlarged, and the triglyceride content was increased. Moreover, the mRNA expression levels of the adipocyte differentiation marker genes, peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein (SREBP), were significantly elevated in the differentiated, mature adipocytes. In contrast, the mRNA expression level of preadipocyte factor 1 (Pref-1) was markedly reduced. E2F transcription factor 2 (E2F2) and Ras-related nuclear (RAN)-binding protein 10 (RANBP10) were the two target genes of miR-378. The mRNA expression levels of E2F2 and RANBP10 did not significantly change in bovine preadipocytes overexpressing miR-378. However, the protein expression levels of E2F2 and RANBP10 were markedly reduced. CONCLUSION: MiR-378 promoted the differentiation of bovine preadipocytes. E2F2 and RANBP10 were the two target genes of miR-378, and might involve in the effects of miR-378 on the bovine preadipocyte differentiation.

Naturally occurring genetic mutations in the 5'-upstream regulatory region of bovine FSHB generate a novel cis-regulatory element that affects its expression.

We previously reported that numerous naturally occurring genetic mutations in the 5'-upstream regulatory region (5'-URR) of the bovine follicle-stimulating hormone beta-subunit gene (FSHB) were associated with reduced serum follicle-stimulating hormone (FSH) levels, poor-quality semen and low fertility in bulls. In addition, two different FSHB mRNA transcripts resulting from the linked mutations of genomic DNA were discovered in mutation-bearing bull pituitaries. Here, using electrophoretic mobility shift assay, we identified c.-1539_-1538delGGinsTTAACT mutations in the 5'-URR that generated a novel cis-regulatory element in bovine FSHB. Moreover, this novel element seemed to play a role in repressing FSHB transcription based on a promoter activity analysis in LßT2 gonadotrope cells. Quantitative assays of FSHB mRNA in the bovine pituitaries suggested that the levels of FSHB wild-type transcripts in the mutation-bearing bulls were significantly lower (P < 0.05) than in those of bulls without FSHB genetic mutations and that the levels of FSHB-mutated transcripts were significantly lower (P < 0.05) than that of wild-type transcripts in the mutation-bearing bulls. Altogether, our results suggest that decreased serum FSH levels and male fertility in bulls with the c.-1539_-1538delGGinsTTAACT mutation likely result from the alteration of cis-regulatory elements and induction of FSHB transcription.

The MBD4 gene plays an important role in porcine adipocyte differentiation.

BACKGROUND: MBD4 (methyl-CpG binding domain protein 4) is an important G: T glycosylase that can identify T-G mismatches. It plays a role in active demethylation through base excision repair. Overexpression of MBD4 gene can cause the demethylation of numerous genes, and the remethylation of MBD4-associated genes can occur when the MBD4 gene is knocked out. To date, the functions and regulatory mechanisms of the MBD4 gene in the differentiation of porcine preadipocytes have not been clearly established. METHODS: Subcutaneous fat cells from 1- to 7-day-old Junmu-1 piglets were cultured in vitro, induced to differentiate, and then identified. A real-time fluorescence-based quantitative polymerase chain reaction (PCR) analysis was conducted to detect MBD4 messenger RNA (mRNA) expression. Cells were treated with MBD4-siRNA (small interfering RNA) and induced to differentiate. Changes in the lipid droplets were observed by oil red O staining. Changes in the mRNA and protein expression levels of MBD4 and the adipose differentiation-associated genes C/EBPα (CCAAT-enhancer-binding protein alpha), PPARγ (peroxisome proliferator-activated receptor gamma), and aP2 (adipocyte protein 2) were detected. In addition, the bisulfite sequencing method was used to detect changes in methylation in the promoters of certain genes associated with adipose differentiation. RESULTS: Levels of MBD4 mRNA and protein expression varied with time over the course of the porcine adipocyte differentiation, with the highest levels of this expression observed on day two of the differentiation process. After silencing MBD4 and inducing differentiation, the production of lipid droplets decreased, the mRNA expression levels of C/EBPα, PPARγ, and aP2 were significantly reduced, and DNA methylation modification levels were significantly elevated in the examined promoter regions. CONCLUSION: The silencing of the MBD4 gene can influence the DNA methylation levels of preadipocyte differentiation-related genes and subsequently inhibit the differentiation of porcine preadipocytes.

Identification of differentially expressed proteins in the ovaries of menopausal women.

PURPOSE: This study investigated proteins differentially expressed in the ovaries of menopausal women in comparison to childbearing women. METHODS: Differential protein expression was screened by difference gel electrophoresis and 2-D SDS-PAGE. Four differentially expressed proteins were excised manually, identified by mass spectrometry and confirmed by immunoblot and immunohistochemistry. RESULTS: The four proteins were identified as serum amyloid P, heat shock protein 27, Glyoxalase I and Ubiquitin carboxy-terminal hydrolase. Serum amyloid P expression was significantly up-regulated in the ovaries of menopausal women by immunoblot analysis (p < 0.05), Glyoxalase I and Ubiquitin carboxy-terminal hydrolase displayed an altered expression pattern, with higher expression in the atretic follicles of menopausal women. Weak Glyoxalase I and Ubiquitin carboxy-terminal hydrolase were observed in the granulosa and theca cells of the follicles of childbearing women. Heat shock protein 27 and serum amyloid P were clearly observed in the atretic follicles of menopausal women, while their expression was restricted to the theca cells and cytoplasm of primordial follicles in the ovaries of childbearing women. All four proteins were predominantly expressed in the atretic follicles of menopausal women. CONCLUSIONS: These data suggest that the identified proteins may play a role in the regulation of follicle atresia in menopausal women, although their functions and mechanism warrant further investigation.

BMP15 prevents cumulus cell apoptosis through CCL2 and FBN1 in porcine ovaries.

BACKGROUND: Bone morphogenetic protein-15 (BMP15) is a maternal gene necessary for mammalian reproduction. BMP15 expression increased in oocytes accompanied by follicle growth and development. The function and regulation mechanism of BMP15 in porcine cumulus cell apoptosis process is still unclear now. METHODS: In this study, flow cytometry (FCM) was used to analyze the effects of BMP15 with different concentrations to cumulus cell apoptosis. High-throughput sequencing technology was carried out to screen regulatory genes linked closely with BMP15. In order to confirm the function of (MCP-1)/CCL2 and FBN1 in cumulus cell apoptosis, RNA interference (RNAi) method was used to inhibit the expression of (MCP-1)/CCL2 and FBN1. Apoptosis and proliferation of cumulus cell treated with siRNA transfection technology were measured by FCM, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, quantitative real time-PCR (RT-qPCR) and western blotting. RESULTS: The results showed that the apoptosis levels of cumulus cell treated by BMP15 decreased significantly in a dose-dependent manner. The expression of related genes protein 1 (MCP-1)/CCL2 and fibrillin1 (FBN1) were both regulated by BMP15. After transfection, the proliferation of porcine cumulus cells increased significantly and apoptosis of cumulus cells was prevented while FBN1 was silenced after BMP15 treatment. The proliferation of cumulus cells decreased significantly and apoptosis rate of cumulus cells increased significantly while CCL2 was silenced. CONCLUSION: The results obtained in this study firstly demonstrated that CCL2 and FBN1 are important regulatory factors of BMP15 in preventing cumulus cell apoptosis in porcine ovaries.

Silencing of ADIPOQ efficiently suppresses preadipocyte differentiation in porcine.

AIMS: Our study aims to characterize the functions of the ADIPOQ gene in the process of fat deposition of pigs, thereby providing a basis for the use of this gene as a molecular marker for pork quality. METHODS: We used healthy Junmu1 piglets less than 7 days of age to establish an in vitro culture system for porcine preadipocytes. Chemically synthesized short hairpin RNAs (shRNA) were transfected into porcine preadipocytes to silence the expression of the ADIPOQ gene. We monitored preadipocyte differentiation and determined the levels of the adipocyte differentiation transcription factors lipoprotein lipase (LPL), peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte fatty acid binding protein (AP2) mRNAs to investigate the effects of ADIPOQ on the differentiation of porcine preadipocytes. RESULTS: After transfection, the mRNA and protein levels of the ADIPOQ gene were significantly decreased (P < 0.01), the number of lipid droplets in the adipocytes was significantly reduced, the OD values reflecting the fat content were significantly decreased (P < 0.01), and the levels of LPL, PPARγ and AP2 were significantly reduced (P < 0.01). CONCLUSIONS: These results suggest that interference with ADIPOQ gene expression can inhibit the differentiation of porcine preadipocytes.

Organoids and metastatic orthotopic mouse model for mismatch repair-deficient colorectal cancer.

Background: Genome integrity is essential for the survival of an organism. DNA mismatch repair (MMR) genes (e.g., MLH1, MSH2, MSH6, and PMS2) play a critical role in the DNA damage response pathway for genome integrity maintenance. Germline mutations of MMR genes can lead to Lynch syndrome or constitutional mismatch repair deficiency syndrome, resulting in an increased lifetime risk of developing cancer characterized by high microsatellite instability (MSI-H) and high mutation burden. Although immunotherapy has been approved for MMR-deficient (MMRd) cancer patients, the overall response rate needs to be improved and other management options are needed. Methods: To better understand the biology of MMRd cancers, elucidate the resistance mechanisms to immune modulation, and develop vaccines and therapeutic testing platforms for this high-risk population, we generated organoids and an orthotopic mouse model from intestine tumors developed in a Msh2-deficient mouse model, and followed with a detailed characterization. Results: The organoids were shown to be of epithelial origin with stem cell features, to have a high frameshift mutation frequency with MSI-H and chromosome instability, and intra- and inter-tumor heterogeneity. An orthotopic model using intra-cecal implantation of tumor fragments derived from organoids showed progressive tumor growth, resulting in the development of adenocarcinomas mixed with mucinous features and distant metastasis in liver and lymph node. Conclusions: The established organoids with characteristics of MSI-H cancers can be used to study MMRd cancer biology. The orthotopic model, with its distant metastasis and expressing frameshift peptides, is suitable for evaluating the efficacy of neoantigen-based vaccines or anticancer drugs in combination with other therapies.

Testis-specific miRNA-469 up-regulated in gonadotropin-regulated testicular RNA helicase (GRTH/DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region: implications of its role in germ cell development.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.

Mesothelioma Mouse Models with Mixed Genomic States of Chromosome and Microsatellite Instability.

Malignant mesothelioma (MMe) is a rare malignancy originating from the linings of the pleural, peritoneal and pericardial cavities. The best-defined risk factor is exposure to carcinogenic mineral fibers (e.g., asbestos). Genomic studies have revealed that the most frequent genetic lesions in human MMe are mutations in tumor suppressor genes. Several genetically engineered mouse models have been generated by introducing the same genetic lesions found in human MMe. However, most of these models require specialized breeding facilities and long-term exposure of mice to asbestos for MMe development. Thus, an alternative model with high tumor penetrance without asbestos is urgently needed. We characterized an orthotopic model using MMe cells derived from Cdkn2a+/-;Nf2+/- mice chronically injected with asbestos. These MMe cells were tumorigenic upon intraperitoneal injection. Moreover, MMe cells showed mixed chromosome and microsatellite instability, supporting the notion that genomic instability is relevant in MMe pathogenesis. In addition, microsatellite markers were detectable in the plasma of tumor-bearing mice, indicating a potential use for early cancer detection and monitoring the effects of interventions. This orthotopic model with rapid development of MMe without asbestos exposure represents genomic instability and specific molecular targets for therapeutic or preventive interventions to enable preclinical proof of concept for the intervention in an immunocompetent setting.

AGO-bound mature miRNAs are oligouridylated by TUTs and subsequently degraded by DIS3L2.

MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3' ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3' binding are sufficient to trigger extensive miRNA 3' modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3' end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.

Cloning and characterization of class 1 and class 2 insulin-like growth factor-I mRNA in Songliao black pig.

Insulin-like growth factor-I (IGF-I) plays a fundamental role in postnatal mammalian growth, development, and metabolism. The mammalian IGF-I gene contains at least six exons from which several different mRNAs are transcribed. In this study, IGF-I mRNA in Songliao black pig liver was investigated using RLM-RACE. Using a 3'-RACE technique, we determined that all mRNA transcripts lacked exon 5 sequence and contained only exon 6 sequence. Using a 5'-RACE technique, we investigated the presence of class 1 and class 2 IGF-I mRNAs. In several other species, the class 1 and class 2 IGF-I polypeptides are generated from mRNAs containing exon 1 or exon 2, respectively. Both class 1 and class 2 IGF-I mRNAs were identified in Songliao black pig liver. Transcription is initiatated upstream of exons 1 and 2 at multiple dispersed start sites to yield two distinct IGF-I mRNA transcript classes which differ in the precursor peptides predicted from their individual leader sequences. Tissue distribution of Songliao black pig class 1 and class 2 IGF-I mRNA was investigated by real-time RT-PCR. Both classes of IGF-I mRNA were expressed in a variety of tissues, however, class 1 IGF-I mRNA was more abundant than class 2 in all tissues.

Structural Differences between Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expand Target Repertoires.

MicroRNA (miRNA) processing begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the maturation of three pri-miR-9 paralogs, which encode the same mature miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its distorted and flexible stem structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Analyses of low-grade glioma patient samples indicate that the alternative-miR-9 has a potential role in tumor progression. Furthermore, we provide evidence that distortion of pri-miRNA stems induced by asymmetric internal loops correlates with Drosha cleavage at non-canonical sites. Our studies reveal that pri-miRNA paralogs can have distinct functions via differential Drosha processing.