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Molecular and Cellular Biology, September 2000, p. 6659-6667, Vol. 20, No. 18
Department of Biochemistry and Molecular
Genetics, University of Colorado Health Sciences Center, Denver,
Colorado 80262,1 and Department of
Biology, Indiana University, Bloomington, Indiana
474052
Received 6 April 2000/Returned for modification 30 May
2000/Accepted 27 June 2000
Genes in Caenorhabditis elegans operons are transcribed
as polycistronic pre-mRNAs in which downstream gene products are
trans spliced to a specialized spliced leader, SL2. SL2 is
donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an
Sm-bound snRNP. SL2 RNA can be conceptually folded into a
phylogenetically conserved three-stem-loop secondary structure. Here we
report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a
substantial loss of trans splicing. Interestingly, the
spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in
trans-splicing efficiency. However, the primary sequence of
stem II is crucial for SL2 trans splicing. Similarly, the
conserved primary sequence of the third stem-loop plays a key role in
trans splicing. While mutations in stem-loop III allow
snRNP formation, a single nucleotide substitution in the loop prevents
trans splicing. In contrast, the analogous region of SL1
RNA is not highly conserved, and its mutation does not abrogate
function. Thus, stem-loop III appears to confer a specific function to
SL2 RNA. Finally, an upstream sequence, previously predicted to be a
proximal sequence element, is shown to be required for SL2 RNA expression.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
trans Splicing of Polycistronic
Caenorhabditis elegans Pre-mRNAs: Analysis of the SL2
RNA
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Genetics, University of Colorado Health
Sciences Center, Denver, CO 80262. Phone: (303) 315-8181. Fax: (303)
315-8215. E-mail: tom.blumenthal{at}UCHSC.edu.
Molecular and Cellular Biology, September 2000, p. 6659-6667, Vol. 20, No. 18
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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