In the sex determination pathway of C. elegans , regulation of the
tra-2 mRNA is essential for female cell fate.
laf-1 regulates translational repression of the
tra-2 mRNA through elements in the
tra-2 3' UTR. The phenotypes of
laf-1 mutants include homozygous embryonic/larval lethality, feminization of some heterozygote, and additional germline defects. We are currently cloning
laf-1 . Based on genetic and SNP mapping
laf-1 has been located to a region of 400 kb between SNP Y71H2B.2 and
daf-2 . Injection of cosmids across this region identified a rescuing fragment of 5 kb. There are no complete predicted open reading frames in this fragment; however, there are at least two predicted Pol III genes. Sequence analysis of these genes suggest that
laf-1 may function as a non-coding RNA. A small fragment of 300 nt containing these two genes produces RNA transcripts in yeast Pol III extracts and Xenopus oocytes consistent with these genes being transcribed by Pol III. We believe one or both of these genes are
laf-1 for the following three reasons: C. elegans
laf-1 mutant strains are partially rescued by the injection of the 300 nt fragment, a mutation has been mapped for one of the
laf-1 alleles that is nine hundred nt upstream of this fragment, and three of the four
laf-1 alleles have been tested in an RNase protection assay and all three show an altered protection pattern compared to wildtype. Primer extension data and RNase protection experiments suggest that the transcript is processed to form the final product. Both the transcription and processing characteristics of this ncRNA indicate that
laf-1 may represent a new class of ncRNA distinct from miRNAs. Sequence based searches have identified a number of other similar predicted Pol III genes in C. elegans, C. briggsae and mouse. At least one of the C. briggsae genes and one of the mouse genes produce transcripts that may be processed in a similar manner to
laf-1 .