The importance of sugar contacts for the sequences-pecific recognition that occurs during polyadenylation of mRNAs was investigated with chemically synthesized substrates containing 2′-OCH, groups at selected riboses. An RNA (5′-CUGCAAUAAACAAGU-UAA-3′) with 2′-OCH, ribose at each nucleotide except for the AAUAAA sequence and 3′-terminal adenosine was efficiently polyadenylated in vitro. Methylation of single riboses within AAUAAA inhibited both poly(A) addition and binding of the specificity factor, but the magnitude of inhibition varied greatly at different nucleotides. Nucleotides that showed sensitivity to base substitutions did not necessarily show sensitivity to ribose methylation, and vice versa. The data indicate that the specificity factor interacts with AAUAAA through RNA-protein contacts involving essential recognition of both sugars and bases at different nucleotide positions.
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We are especially grateful to Dr. Barbro Beijer for provision of 2'-O-FPMP-protected ribonucleoside phosphoramidites. We thank Dave Zarkower for providing crude fractions of HeLa cell nuclear extract. We appreciate Elmar Wahla's generous gift of poly(A) polymerase and his helpful advice and criticism during the work. We are grateful to Professor Lennart Philipson and Drs. Richard Treisman, lain Mattaj, Mathias Hentze, Henk Stunnenberg, Giovanni Paolella, Ben Blen-cowe, and Silvia Barabino for making constructive criticisms of the manuscript. We thank Laura Conway for help during the early phase of these experiments and Vince Schulz for advice on alkali hydrolysis. We appreciate the help of Philippe Neunerwith oligonucleotide synthesis. Laura Vanderploeg provided excellent technical assistance in the preparation of figures. V. J. B. was supported by a Natural Sciences and Engineering Research Council of Canada postgraduate fellowship. This work was in part supported by the following grants: Research Grant (GM31892) and Research Career Development Award (GM00521) from the National Institutes of Health to M. W., grants from the Schweiz-erischer Nationalfonds, and the Kantons of Basel to W. K. V. J. B. was also the recipient of an EMBO short-term fellowship during the latter stages of this work.