LIN41 Post-transcriptionally Silences mRNAs by Two Distinct and Position-Dependent Mechanisms.

The RNA-binding protein (RBP) LIN41, also known as LIN-41 or TRIM71, is a key regulator of animal development, but its physiological targets and molecular mechanism of action are largely elusive. Here we find that this RBP has two distinct mRNA-silencing activities. Using genome-wide ribosome profiling, RNA immunoprecipitation, and in vitro-binding experiments, we identify four mRNAs, each encoding a transcription factor or cofactor, as direct physiological targets of C. elegans LIN41. LIN41 silences three of these targets through their 3' UTRs, but it achieves isoform-specific silencing of one target, lin-29A, through its unique 5' UTR. Whereas the 3' UTR targets mab-10, mab-3, and dmd-3 undergo transcript degradation, lin-29A experiences translational repression. Through binding site transplantation experiments, we demonstrate that it is the location of the LIN41-binding site that specifies the silencing mechanism. Such position-dependent dual activity may, when studied more systematically, emerge as a feature shared by other RBPs.

A genetic interactome of the let-7 microRNA in C. elegans.

The heterochronic pathway controls temporal patterning during Caenorhabditis elegans larval development. The highly conserved let-7 microRNA (miRNA) plays a key role in this pathway, directing the larval-to-adult (L/A) transition. Hence, knowledge of the genetic interactome of let-7 has the potential to provide insight into both control of temporal cell fates and mechanisms of regulation and function of miRNAs. Here, we report the results of a genome-wide, RNAi-based screen for suppressors of let-7 mutant vulval bursting. The 201 genetic interaction partners of let-7 thus identified include genes that promote target silencing activity of let-7, seam cell differentiation, or both. We illustrate the suitability of our approach by uncovering the mitotic cyclin-dependent kinase CDK-1 as a downstream effector of let-7 that affects both seam cell proliferation and differentiation, and by identifying a core set of candidate modulators of let-7 activity, which includes all subunits of the condensin II complex. We propose that the genes identified in our screen thus constitute a valuable resource for studies of the heterochronic pathway and miRNAs.

Amniocytes can serve a dual function as a source of iPS cells and feeder layers.

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5-7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.