ISOLATION OF RRM-TYPE RNA-BINDING PROTEIN GENES AND THE ANALYSIS OF THEIR RELATEDNESS BY USING A NUMERICAL APPROACH

Proteins with RNA recognition motifs (RRMs) have important roles in a great many aspects of RNA metabolism. However, this family has yet to be systematically studied in any single organism. In order to investigate the size of the RRM gene family in Drosophila melanogaster and to clone members of this family, we used a polymerase chain reaction (PCR) with highly degenerate oligonucleotides to amplify DNA fragments between the RNP-1 and RNP-2 consensus sequences of the RRM proteins. Cloning and sequencing of 124 PCR products revealed 12 different RRM sequences (RRM1 to RRM12). When PCR products were used as probes in genomic Southern and Northern (RNA) analyses, 16 restriction fragments and 25 transcripts, respectively, were detected. Since the combinations of nucleotide sequences represented in the PCR primers correspond to only 4% of the RRM sequences inferred to be possible from known RRM sequences, we estimate the size of the RRM gene family in the order of three hundred genes in flies. In order to gain insight into the possible functions of the genes encoding the RRMs, we analyzed the sequence similarities between the 12 RRMs and 62 RRM sequences of known proteins. This analysis showed that the RRMs of functionally related proteins have similar sequences and are clustered together in the RRM gene tree. On the basis of this observation, the RRMs can be divided into three groups: a heterogeneous nuclear ribonucleoprotein type, a splicing regulator type, and a development-specific factor type. This result suggests that we have isolated good candidates for both housekeeping and developmentally important genes involved in RNA metabolism.