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Molecular and Cellular Biology, April 1999, p. 2782-2790, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

Véronique Ségault,1 Cindy L. Will,2 Maria Polycarpou-Schwarz,3 Iain W. Mattaj,3 Christiane Branlant,1 and Reinhard Lührmann2,*

UMR CNRS 7567 Maturation des ARN et Enzymologie Moleculaire Université H. Poincaré, 54506 Vandoeuvre-Les-Nancy Cédex, France,1 and Institut für Molekularbiologie und Tumorforschung, Philipps Universität Marburg, 35037 Marburg,2 and EMBL, 69117 Heidelberg,3 Germany

Received 2 November 1998/Returned for modification 1 December 1998/Accepted 28 December 1998

The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa or Xenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5' and 3' halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

* Corresponding author. Mailing address: Institut für Molekularbiologie und Tumorforschung, Emil-Mannkopff Str. 2, D-35037 Marburg, Germany. Phone: 49-6421-286240. Fax: 49-6421-287008. E-mail: luehrmann{at}imt.uni-marburg.de.

Molecular and Cellular Biology, April 1999, p. 2782-2790, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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