Molecular Biology Reports 14: 143, 1990. 9 1990 Kluwer Academic Publishers. Printed in Belgium.
CONTRIBUTION
OF U1 snRNA STRUCTURAL
143
DOMAINS
TO U1 snRNP FUNCTION
XIAOLING LIAO, LEO KRETZNER*, BERTRAND SERAPHIN + and M I C H A E L ROSBASH Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, Massachusetts, 02254, USA Current address: Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington, 98104, USA +Current address: Institute Curie, Section de Biologie, Batiment ii0, Campus Universitaire, D-91405 Orsay Cedex, France
Based on p h y l o g e n e t i c comparisons and structure probing, we have derived a secondary structure model of the large yeast ( S a c c h a r o m y c e s c e r e v i s a e ) U1 snRNA. The model contains most secondary structure and several primary structure features of m e t a z o a n U1 snRNA, including the stem I and the highly conserved A loop. In addition, the data suggest that at least a subset of the highly structured y e a s t - s p e c i f i c regions are functional because they are w e l l - c o n s e r v e d between d i f f e r e n t yeast species. I To address functional issues, we have made complete deletions of the A loop and of the most highly conserved yeast specific region (the yeast core), r e i n t r o d u c e d these molecules into yeast by gene replacement techniques, and analyzed their phenotypes. The strain that carries the A loop deletion has no d i s c e r n a b l e growth phenotype. The strain that carries the yeast core d e l e t i o n is t e m p e r a t u r e - s e n s i t i v e and extracts derived from this strain are defective; they m a n i f e s t a block after commitment complex formation, i.e., after formation of a U l : p r e - m R N A complex. Double mutant m o l e c u l e s (that carry both the yeast core deletion and the A loop deletion) are lethal, indicating that the
conserved A loop does contribute to function, but that this contribution is invisible when the deletion is imbedded in an otherwise w i l d - t y p e U1 molecule. The implications of these results for U1 snRNP function and p r e - m R N A splicing will be discussed.
REFERENCE
I. Kretzner, L., Krol, A. and Rosbash, M. Saccharomycescerevisiae U1 small nuclear RNA secondary structure contains both universal and y e a s t - s p e c i f i c domains. Proc. Natl. Acad. Sci. USA. In press (1990).
CONTRIBUTION
OF U1 snRNA STRUCTURAL
143
DOMAINS
TO U1 snRNP FUNCTION
XIAOLING LIAO, LEO KRETZNER*, BERTRAND SERAPHIN + and M I C H A E L ROSBASH Howard Hughes Medical Institute and Department of Biology, Brandeis University, Waltham, Massachusetts, 02254, USA Current address: Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington, 98104, USA +Current address: Institute Curie, Section de Biologie, Batiment ii0, Campus Universitaire, D-91405 Orsay Cedex, France
Based on p h y l o g e n e t i c comparisons and structure probing, we have derived a secondary structure model of the large yeast ( S a c c h a r o m y c e s c e r e v i s a e ) U1 snRNA. The model contains most secondary structure and several primary structure features of m e t a z o a n U1 snRNA, including the stem I and the highly conserved A loop. In addition, the data suggest that at least a subset of the highly structured y e a s t - s p e c i f i c regions are functional because they are w e l l - c o n s e r v e d between d i f f e r e n t yeast species. I To address functional issues, we have made complete deletions of the A loop and of the most highly conserved yeast specific region (the yeast core), r e i n t r o d u c e d these molecules into yeast by gene replacement techniques, and analyzed their phenotypes. The strain that carries the A loop deletion has no d i s c e r n a b l e growth phenotype. The strain that carries the yeast core d e l e t i o n is t e m p e r a t u r e - s e n s i t i v e and extracts derived from this strain are defective; they m a n i f e s t a block after commitment complex formation, i.e., after formation of a U l : p r e - m R N A complex. Double mutant m o l e c u l e s (that carry both the yeast core deletion and the A loop deletion) are lethal, indicating that the
conserved A loop does contribute to function, but that this contribution is invisible when the deletion is imbedded in an otherwise w i l d - t y p e U1 molecule. The implications of these results for U1 snRNP function and p r e - m R N A splicing will be discussed.
REFERENCE
I. Kretzner, L., Krol, A. and Rosbash, M. Saccharomycescerevisiae U1 small nuclear RNA secondary structure contains both universal and y e a s t - s p e c i f i c domains. Proc. Natl. Acad. Sci. USA. In press (1990).
