MicroRNA-Biogenesis and Pre-mRNA Splicing Crosstalk

MicroRNAs (miRNAs) are often hosted in introns of protein-coding genes. Given that the same transcriptional unit can potentially give rise to both miRNA and mRNA transcripts raises the intriguing question of the level of interaction between these processes. Recent studies from transcription, pre-mRNA splicing, and miRNA-processing perspectives have investigated these relationships and yielded interesting, yet somewhat controversial findings. Here we discuss major studies in the field

Human disease locus discovery and mapping to molecular pathways through phylogenetic profiling

Genes with common profiles of the presence and absence in disparate genomes tend to function in the same pathway. By mapping all human genes into about 1000 clusters of genes with similar patterns of conservation across eukaryotic phylogeny, we determined that sets of genes associated with particular diseases have similar phylogenetic profiles. By focusing on those human phylogenetic gene clusters that significantly overlap some of the thousands of human gene sets defined by their coexpression or annotation to pathways or other molecular attributes, we reveal the evolutionary map that connects molecular pathways and human diseases. The other genes in the phylogenetic clusters enriched for particular known disease genes or molecular pathways identify candidate genes for roles in those same disorders and pathways. Focusing on proteins coevolved with the microphthalmia-associated transcription factor (MITF), we identified the Notch pathway suppressor of hairless (RBP-Jk/SuH) transcription factor, and showed that RBP-Jk functions as an MITF cofactor

Analysing human neural stem cell ontogeny by consecutive isolation of Notch active neural progenitors

Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease

Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs

Interplay between MITF, PIAS3, and STAT3 in Mast Cells and Melanocytes

Microphthalmia transcription factor (MITF) and STAT3 are two transcription factors that play a major role in the regulation of growth and function in mast cells and melanocytes. In the present study, we explored the MITF-PIAS3-STAT3 network of interactions, how these interactions regulate gene expression, and how cytokine-mediated phosphorylation of MITF and STAT3 is involved in the in vivo interplay between these three proteins. In NIH 3T3 cells stimulated via gp130 receptor, transfected MITF was found to be phosphorylated at S409. Such phosphorylation of MITF leads to PIAS3 dissociation from MITF and its association with STAT3. Activation of mouse melanoma and mast cells through gp130 or c-Kit receptors induced the mobilization of PIAS3 from MITF to STAT3. In mast cells derived from MITF(di/di) mice, whose MITF lacks the Zip domain (PIAS3-binding domain), we found downregulation in mRNA levels of genes regulated by either MITF or STAT3. This regulatory mechanism is of considerable importance since it is likely to advance the deciphering of a role for MITF and STAT3 in mast cells and melanocytes

Role Played by Microphthalmia Transcription Factor Phosphorylation and Its Zip Domain in Its Transcriptional Inhibition by PIAS3

Mutation of microphthalmia transcription factor (MITF) results in deafness, bone loss, small eyes, and poorly pigmented eyes and skin. A search for MITF-associated proteins, using a mast cell library that was screened with a construct that encodes the basic helix-loop-helix leucine zipper (Zip) domain of MITF, resulted in the isolation of the STAT3 inhibitor, PIAS3. PIAS3 functions in vivo as a key molecule in suppressing the transcriptional activity of MITF. Here, we report that the Zip domain is the region of MITF that is involved in the direct interaction between MITF and PIAS3. Additionally, we investigated the effect of phosphorylation of MITF on its interaction with PIAS3. We found that phosphorylation of MITF on serines in positions 73 and 409 plays an important role in its association with PIAS3. This effect was profound with phosphorylation on Ser409, which significantly reduced the inhibitory effect of PIAS3 on MITF and also modulated the transcriptional activity of MITF. Thus, phosphorylation of MITF could be considered a fine, and alternative, tuning of its transcriptional machinery