MicroRNAs (miRNAs) are noncoding RNAs of approximately 22 nucleotides in length that negatively regulate the post-transcriptional expression by translational repression and/or destabilization of protein-coding mRNAs. The impact of miRNAs on protein output was recently shown that although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. Thus, comparative profilings of miRNAs and mRNAs from the same samples of different cell types may identify the putative targets of miRNAs. In this investigation, both miRNA and mRNA profiles from the undifferentiated human embryonic stem cell line hES-T3 (T3ES), hES-T3 derived embryoid bodies (T3EB), and hES-T3 differentiated fibroblast-like cells (T3DF) were compared, and 58 genes were found to be targets of four hES cell-specific miRNAs miR-302d, miR-372, miR-200c and/or miR-367 by inverse expression levels (highly negative correlation) of miRNAs to their target mRNAs. Approximately half of these 58 targets are involved in gene transcription. Three common target genes TRPS1, KLF13 and MBNL2 of three highly expressed miRNAs miR-302d, miR-372, and miR-200c were identified, and the target sites of both miR-302d and miR-372 in the 3 0 UTR of TRPS1, KLF13, and MBNL2 genes were confirmed by the Journal of Cellular Biochemistry ARTICLE

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A total of 18 families with multiple cases of breast cancer were identified from southern Taiwan, and 5 of these families were found to carry cancer-associated germline mutations in the BRCA1 and BRCA2 genes. One novel cryptic splicing mutation of the BRCA1 gene, found in two unrelated families, was shown to be a deletion of 10 bp near the branch site in intron 7. This mutation causes an insertion of 59 nucleotides derived from intron 7 and results in a frameshift, leading to premature translational termination of BRCA1 mRNA in exon 8. Deletions of 2670delC, 3073delT and 6696-7delTC in the BRCA2 gene were found in three other breast cancer families. All three deletions are predicted to generate frameshifts and to result in the premature termination of BRCA2 protein translation. Several genetic polymorphisms in both BRCA1 and BRCA2 genes were also detected in this investigation.

Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self-renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES-T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet-like cell clusters derived from T3 cells), which expressed pancreatic islet cell-specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES-T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein-coding mRNAs. The T3pi showed very high expression of microRNAs, miR-186, miR-199a and miR-339, which down-regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics.

Protein sumoylation by small ubiquitin-like modifier (SUMO) proteins is an important post-translational regulatory modification. A role in the control of chromosome dynamics was first suggested when SUMO was identified as high-copy suppressor of the centromere protein CENP-C mutants. CENP-C itself contains a consensus sumoylation sequence motif that partially overlaps with its DNA binding and centromere localization domain. To ascertain whether CENP-C can be sumoylated, tandem mass spectrometry (MS) based strategy was developed for high sensitivity identification and sequencing of sumoylated isopeptides present among in-gel-digested tryptic peptides of SDS-PAGE fractionated target proteins. Without a predisposition to searching for the expected isopeptides based on calculated molecular mass and relying instead on the characteristic MS/MS fragmentation pattern to identify sumolylation, we demonstrate that several other lysine residues located not within the perfect consensus sumoylation motif KXE/D, where represents a large hydrophobic amino acid, and X represnts any amino acid, can be sumolylated with a reconstituted in vitro system containing only the SUMO proteins, E1-activating enzyme and E2-conjugating enzyme (Ubc9). In all cases, target sites that can be sumoylated by SUMO-2 were shown to be equally susceptible to SUMO-1 attachments which include specific sites on SUMO-2 itself, Ubc9, and the recombinant CENP-C fragments. Two non-consensus sites on one of the CENP-C fragments were found to be sumoylated in addition to the predicted site on the other fragment. The developed methodologies should facilitate future studies in delineating the dynamics and substrate specificities of SUMO-1/2/3 modifications and the respective roles of E3 ligases in the process.Protein modification by covalent attachment of SUMO 1 (small ubiquitin-like modifier) proteins is emerging as an important regulatory mechanism in a diverse range of cellular processes (1, 2). Through a three-step enzymatic pathway analogous to ubiquitylation, sumoylation is initiated by ATP-dependent formation of a thioester bond between the C-terminal glycine of a SUMO protein and a catalytic cysteine of the SUMO-specific E1-activating enzyme, known as SAE1/SAE2 and Uba2p/Aos1p for human and yeast, respectively. The activated SUMO protein is then trans-esterified from the E1 enzyme to the catalytic cysteine of an E2-conjugating enzyme, Ubc9, which in turn catalyzes the formation of an isopeptide bond between the C-terminal glycine of SUMO and the ⑀-amino group of lysine residue in the substrate proteins. Ubc9 was reported to recognize and bind directly to a consensus sumoylation motif KXE/D, where represents a large hydrophobic amino acid L, I, V, or F, and X represents any amino acid (3-5). Although sumoylation can be accomplished in vitro without an E3-like enzyme, several E3 ligases, e.g. PIAS, RanBP2, and PC2, have been identified which may aid Ubc9 in substrate selection and ligation efficiency in vivo (2, 6).Unlike yeast and other invertebrates, whi...