Dataset Information

MonoMac6 (MM6) cells proteomics LC-MS/MS

ABSTRACT: MicroRNAs (miRs) are small non-coding RNAs, which control the expression of target genes by either translational repression or RNA degradation, known as canonical miR function. MiR-328 has a non-canonical function and can activate gene expression by antagonizing the activity of heterogeneous ribonuclear protein E2 (hnRNP E2). The global importance of such non-canonical miR function is not yet known. In order to achieve a better understanding of the non-canonical miR activity, a compartment specific tandem mass tag (TMT)-based proteomic analysis in differentiated MonoMac6 (MM6) cells was performed.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Culture

SUBMITTER: Elena Ossipova

LAB HEAD: Per-Johan Jakobsson

PROVIDER: PXD013106 | Pride | 2019-11-27

REPOSITORIES: Pride

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Publications

Mass Spectrometry-Based Proteomics Approach Characterizes the Dual Functionality of miR-328 in Monocytes.

Saul Meike J MJ Hegewald Anett B AB Emmerich Anne C AC Ossipova Elena E Vogel Marc M Baumann Isabell I Kultima Kim K Lengqivst Johan J Steinhilber Dieter D Jakobsson Per Johan PJ

Frontiers in pharmacology 20190605

MicroRNAs (miRs) are small noncoding RNAs which control the expression of target genes by either translational repression or RNA degradation, known as canonical miR functions. The recent discovery that miR-328 has a noncanonical function and can activate gene expression by antagonizing the activity of heterogeneous ribonuclear protein E2 (hnRNP E2) opens an unexplored and exciting field of gene expression regulation. The global importance of such noncanonical miR function is not yet known. In or ...[more]

PMID: 31231226

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Project description:Modification of microRNA sequences by the 3' addition of nucleotides to generate so-called M-bM-^@M-^\isomiRsM-bM-^@M-^] adds to the complexity of miRNA function, with recent reports showing that 3' modifications can influence miRNA stability and efficiency of target repression. Here we show that the 3' modification of miRNAs is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species ranging from C. elegans to human. The modifications result predominantly from adenylation and uridylation, and are seen across tissue types, disease states, and developmental stages. To quantitatively profile 3' nucleotide additions, we developed and validated a novel assay based on NanoString Technologies' nCounter platform. For certain miRNAs, the frequency of modification was altered by processes such as cell differentiation, indicating that 3' modification is a biologically regulated process. To investigate the mechanism of 3' nucleotide additions, we used RNA interference to screen a panel of eight candidate miRNA nucleotidyl transferases for 3' miRNA modification activity in human cells. Multiple enzymes, including PAPD1, PAPD4, PAPD5, ZCCHC6, ZCCHC11, and TUT1, were found to govern 3' nucleotide addition to miRNAs in a miRNA-specific manner. Three of these enzymesM-bM-^@M-^SPAPD1, ZCCHC6 and TUT1M-bM-^@M-^Shave not previously been known to modify miRNAs. Collectively, our results indicate that 3' modification observed in next generation small RNA sequencing data is a biologically relevant process, and identify enzymatic mechanisms that may lead to new approaches for modulating miRNA activity in vivo. We validated the specificity of the platform by assaying three pools of synthetic RNA oligonucleotides representing canonical, variant 1, and variant 2 versions of 5 miRNAs (miR-15a, miR-15b, miR-125a-5p, miR-143 and miR-221).Each bridge pool (i.e., specific to canonical, variant 1 or variant 2) was used to assay the three mixtures of synthetic miRNA oligonucleotides. In order to assess accuracy, we also assayed mixtures of 5 synthetic miRNAs containing 60% canonical miRNA, 30% variant 1, and 10% variant 2. Triplicate technical replicates of each sample were performed.

Dataset Information

MonoMac6 (MM6) cells proteomics LC-MS/MS

Publications

Mass Spectrometry-Based Proteomics Approach Characterizes the Dual Functionality of miR-328 in Monocytes.

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