An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans
Tóm tắt
Two small temporal RNAs (stRNAs),
Từ khóa
Tài liệu tham khảo
Short endogenous C. elegans RNAs were cloned using a protocol inspired by Elbashir et al. (12) but modified to make it specific for RNAs with 5′-terminal phosphate and 3′-terminal hydroxyl groups. In our protocol (24) gel-purified 18–26 nt RNA from mixed-stage worms was ligated to a pre-adenylylated 3′-adaptor oligonucleotide in a reaction using T4 RNA ligase but without adenosine triphosphate (ATP). Ligated RNA was gel-purified then ligated to a 5′-adaptor oligonucleotide in a standard T4 RNA ligase reaction. Products from the second ligation were gel-purified then reverse transcribed and amplified by using the primers corresponding to the adaptor sequences. To achieve ligation specificity for RNA with a 5′-terminal phosphate and 3′-terminal hydroxyl phosphatase and phosphorylase treatments useful for preventing circularization of Dicer products (12) were not included in our protocol. Instead circularization was avoided by using the pre-adenylylated 3′-adaptor oligonucleotide and omitting ATP during the first ligation reaction.
Supplemental material describing methods and predicted fold-back secondary structures for the miRNAs of Table 1 and some of their homologs in other species is available on Science Online at www.sciencemag.org/cgi/content/full/294/5543/858/DC1.
N. C. Lau L. P. Lim E. Weinstein D. P. Bartel data not shown.
This begs the question as to why more riboregulators have not been found previously. Perhaps they had not been identified biochemically because of a predisposition towards searching for protein rather than RNA factors. They could be identified genetically which was how lin-4 and let-7 were discovered (1–3); however when compared to mutations in protein-coding genes point substitutions in these short RNA genes would be less likely and perhaps less disruptive of function. Furthermore mutations that map to presumed intergenic regions with no associated RNA transcript detectable on a standard RNA blot might be put aside in favor of other mutants.
WormBase is available on the Web at www.wormbase.org.
Sequencing traces (from the Sanger Center) representing 2.5- to 3-fold average coverage of the C. briggsae genome were obtained at www.ncbi.nlm.nih.gov/Traces.
We thank C. Ceol for guidance in culturing and staging C. elegans ; R. Horvitz for the use of equipment and facilities; T. Tuschl G. Ruvkun B. Reinhart A. Pasquinelli C. Burge F. Lewitter A. Ensminger and C. Mello for helpful discussions; P. Zamore T. Orr-Weaver and M. Lawrence for comments on this manuscript; and T. Tuschl V. Ambros G. Ruvkin R. Horvitz P. Sharp and J. Hodgkin for discussions on nomenclature.