Project description:Recent small RNA sequencing data has uncovered extensive modification of the 3’ end of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3’ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Hox gene control. Zcchc6/11 selectively uridylate these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3’ mono-uridylation of many of the same miRNAs. Interestingly, upon TUTase dependent loss of uridylation we observe a concomitant increase in non-templated 3’ mono-adenylation. Our results uncover the molecular basis for sequence specific miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3’ miRNA modifications in cells, and have implications for miRNA regulation during development.
Project description:Recent small RNA sequencing data has uncovered extensive modification of the 3’ end of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3’ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Hox gene control. Zcchc6/11 selectively uridylate these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3’ mono-uridylation of many of the same miRNAs. Interestingly, upon TUTase dependent loss of uridylation we observe a concomitant increase in non-templated 3’ mono-adenylation. Our results uncover the molecular basis for sequence specific miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3’ miRNA modifications in cells, and have implications for miRNA regulation during development. small RNA profiles in TUTases knock-down and control HeLa cells were generated by Illumina deep sequencing
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:The generation of pancreatic organoids from human pluripotent stem cells represents a major breakthrough for regenerative medicine and the modeling of diseases such as diabetes. However, current approaches remain inefficient due to lengthy multi-step differentiation protocols and limited functional maturity in the organoids. In this study, we overcome these challenges using multi-phase optimization screens to achieve rapid generation of functionally mature pancreatic organoids from a stable endocrine progenitor culture. We conducted stepwise culture condition screens that enabled the stable culture of multiple pancreatic progenitor cell states, including the unprecedented stable propagation of NEUROD1-expressing endocrine progenitor-like cells (EpSCs). Further transcriptomic profiling of EpSC confirmed similarity of that to previously reported endocrine progenitor populations. Using EpSCs, we significantly reduced the number of steps and timing required to generate pancreatic organoids, enabling rapid testing of conditions for organoid maturation. Utilizing this optimized protocol, we further tested conditions to promote pancreatic organoid maturation. We identified that exosome-delivered WNT5B, in combination with RSPO1 (exoW/R), could strongly induce non-canonical WNT/JNK signaling, promoting pancreatic organoid maturation. This combinatorial exosome treatment enhances epithelial organization, reduces immature cell states, and significantly improves glucose responsiveness and insulin secretion. Collectively, our work establishes a robust pancreatic differentiation platform that integrates long-term progenitor expansion with optimized organoid maturation. This system provides a reproducible experimental framework for studying pancreatic development, investigating disease mechanisms, and facilitating future translational applications involving pancreatic organoids.
Project description: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 have 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 for uridylated isomiRs in regulating non-canonical miRNA targets.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.