hnRNPC induces isoform shifts in miR-21-5p leading to cancer development.

MicroRNA (miRNA) processing is a critical step in mature miRNA production. Its dysregulation leads to an increase in miRNA isoforms with heterogenous 5'-ends (isomiRs), which can recognize distinct target sites because of their shifted seed sequence. Although some miRNA genes display productive expression of their 5'-isomiRs in cancers, how their production is controlled and how 5'-isomiRs affect tumor progression have yet to be explored. In this study, based on integrative analyses of high-throughput sequencing data produced by our group and publicly available data, we demonstrate that primary miR-21 (pri-miR-21) is processed into the cancer-specific isomiR isomiR-21-5p | ±1, which suppresses growth hormone receptor (GHR) in liver cancer. Treatment with antagomirs against isomiR-21-5p | ±1 inhibited the in vitro tumorigenesis of liver cancer cells and allowed the recovery of GHR, whereas the introduction of isomiR-21-5p | ±1 mimics attenuated these effects. These effects were validated in a mouse model of spontaneous liver cancer. Heterogeneous nuclear ribonucleoprotein C and U2 small nuclear RNA auxiliary factor 2 were predicted to bind upstream of pre-miR-21 via a poly-(U) motif and influence Drosha processing to induce the production of isomiR-21-5p | ±1. Our findings suggest an oncogenic function for the non-canonical isomiR-21-5p | ±1 in liver cancer, and its production was shown to be regulated by hnRNPC.

UPF1/SMG7-dependent microRNA-mediated gene regulation.

The stability and quality of metazoan mRNAs are under microRNA (miRNA)-mediated and nonsense-mediated control. Although UPF1, a core mediator of nonsense-mediated mRNA decay (NMD), mediates the decay of target mRNA in a 3'UTR-length-dependent manner, the detailed mechanism remains unclear. Here, we suggest that 3'UTR-length-dependent mRNA decay is not mediated by nonsense mRNAs but rather by miRNAs that downregulate target mRNAs via Ago-associated UPF1/SMG7. Global analyses of mRNAs in response to UPF1 RNA interference in miRNA-deficient cells reveal that 3'UTR-length-dependent mRNA decay by UPF1 requires canonical miRNA targeting. The destabilization of miRNA targets is accomplished by the combination of Ago2 and UPF1/SMG7, which may recruit the CCR4-NOT deadenylase complex. Indeed, loss of the SMG7-deadenylase complex interaction increases the levels of transcripts regulated by UPF1-SMG7. This UPF1/SMG7-dependent miRNA-mediated mRNA decay pathway may enable miRNA targeting to become more predictable and expand the miRNA-mRNA regulatory network.

PCHM: A bioinformatic resource for high-throughput human mitochondrial proteome searching and comparison.

Mitochondrial proteins associated with a wide spectrum of human diseases and currently large amounts of tissue or organ specific human mitochondrial proteome datasets are generated. However, high-throughput comparative proteomic methods have yet to be applied to extract subtle differences among mitochondria from different tissues or muscle types. The aim of this work was to provide an integrated way to identify and compare huge mitochondrial protein or peptide mass spectral data sets acquired from expert mitochondrial proteome or biomarker discovery community. Proteome comparison of human mitochondria (PCHM) is a web-based analysis environment for manual or automatic analysis of individual peptide mass fingerprints alongside a database of proteins and peptides identified in various organs for human mitochondrial proteins. PCHM provides a suite of graphical tools that allow the virtual plot of peptide mass fingerprinting (PMF) spectra and a fully automatic protein function classification based on gene ontology (GO) annotation system. The new virtual PMF plot is very useful to validate fragmented ion loses of any identical proteins and to remove unwanted foreign ion peaks. Fully automatic protein function classifier provides an easier way to compare the subtle differences of compositionally biased mitochondrial protein functions. PCHM also provides a variety of query algorithms aid in browsing, searching, and accessing complete annotations of data relevant to each mitochondrial protein of interest, which link external databases and users. PCHM will be a useful tool for the systematic and functional characterization of the mitochondrial proteins in relation to human diseases or biological research applications. PCHM can be accessed freely via a web interface http://pchm.inje.ac.kr.

Generation of monoclonal antibody recognized by the GXXXG motif (glycine zipper) of prion protein.

To develop monoclonal antibodies (MAbs) to react with normal prion protein (PrPC) and abnormal isoform of prion protein (PrPSc), PrPSc was isolated from brains of 263 K scrapie-infected hamsters and immunized to PrP knockout mice. We developed two hybridomas, 3F10 and 1C5 (IgG1), of which epitope mappings were screened by using glutathione S-transferase (GST) fusion proteins of recombinant hamster prion protein and suitable peptides. 3F10 showed a high affinity for hamster and mouse PrP and was demonstrated to recognize the residues 137-151. 1C5 recognizes the region 119-130 corresponding to the GXXXG motif, the glycine zipper region, conserved in all mammals. In the immunohistochemical analysis, the positive staining for PrPSc was observed in the extracellular compartment of scrapie-infected brains but not in the normal brains. However, in Western blot, these antibodies recognized both normal and abnormal prion proteins. These results suggested that the developed mouse MAbs are specific to prion protein and can recognize abnormal prion protein more effectively than normal prion protein in immunohistochemistry. Therefore, these antibodies could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties between PrPC and PrPSc.