The identification of the large class of miRNAs in plants, vertebrates, and invertebrates, as well as, the characterization of many ncRNAs in bacteria has revolutionized how we think of ncRNAs in biology. Do other classes exist? Identification and characterization of other families of ncRNAs is essential for understanding how general is ncRNA based control. C. elegans hermaphrodites are somatically female; however, the germline produces sperm then oocytes. Hermaphrodite spermatogenesis requires the translational repression of tra-2 by two 3'untranslated elements (called TGEs). laf-1 acts through the TGEs to inhibit tra-2 activity. It is also an essential gene suggesting it regulates other mRNAs besides tra-2. We have cloned laf-1 and find it encodes a not previously described type ncRNA that appears to be produced by the splicing of a precursor Pol lII transcript. We have identified similar ncRNAs in C. briggsae and mouse indicating this class of ncRNAs is broadly conserved. In the next five years, we propose to investigate the expression and processing of this class of ncRNAs. We will use genetics, transgenes and molecular biology to ask what are the cis-acting elements and trans-acting factors required for splicing. Moreover, we will use transgenes and molecular biology to investigate how TRA-1 influences laf-] metabolism. Our data finds that TRA-1 affects the size of the laf-1 ncRNA, suggesting it is required for expression, processing, or stability of laf-1. In addition, we will use RNAi, RNA in situ and molecular analyses to ask how laf-1 interacts with the Wnt and SRC-1 pathways. Preliminary data is consistent with laf-1 acting downstream, upstream or in parallel to the Wnt and/or SRC-1 pathways to control germline sexual development and spindle orientation of the ABar blastomere in the early embryo. We will also screen for suppressors of laf-1 to identify genes required for expression and processing, as well as, potential laf-1 targets. We have already identified a mutation that suppresses the laf-1 lethality. We will clone and characterize this gene. In addition, we will continue to screen for more laf-1 supressors. Using computational and molecular biology, we will look for other members of this ncRNA family. The health relatedness of this work derives from its contribution to an understanding of fundamental control of gene expression. Understanding how this family of ncRNAs is expressed and processed may result in the ability to engineer ncRNAs that could regulate mRNAs of ones choosing, which would have health care applications.
| Hubert, Amy; Anderson, Philip (2009) The C. elegans sex determination gene laf-1 encodes a putative DEAD-box RNA helicase. Dev Biol 330:358-67 |
