THE HUMAN Y4 SMALL CYTOPLASMIC RNA GENE IS CONTROLLED BY UPSTREAM ELEMENTS AND RESIDES ON CHROMOSOME-7 WITH ALL OTHER HY SCRNA GENES

Ro ribonucleoproteins (RNP) constitute a class of evolutionarily conse rved small cytoplasmic (sc) RNPs whose functions are unknown. In human cells four distinctive scRNAs designated hY1, hY3, hY4 and hY5 are sy nthesized by RNA polymerase III (poi III) and accumulate as components of Ro scRNPs. The previously isolated hY1 and hY3 genes contain upstr eam sequences similar to the class III promoters for Us and 7SK snRNAs . Additional mammalian Y scRNA genes have been refractory to cloning d ue to interference from numerous hY-homologous pseudogenes and studies of hY RNA genes have been sparse. Although homologs of hY1 and hY3 RN As exist in rodent cells, the smaller Y4 and Y5 RNAs do not which has allowed us to localize the hY4 scRNA gene to human chromosome 7 by ass aying for its transcript in rodent X human somatic cell hybrids (SCH). A chromosome 7-enriched yeast artificial chromosome (YAC) library was then screened and the authentic hY4 sequence was isolated by strepavi din - biotin-mediated hybrid-selection followed by poly(dA)-tailing an d hemispecific PCR. The region upstream of the hY4 sequence contains a TATAAAA motif centered at - 26, a candidate proximal sequence element at - 63, and three octamer-like sequences located between - 260 and - 200. hY4 RNA is readily detectable on Northern blots after transient transfection of the hY4 gene into mouse cells but not after transfecti on of a construct in which the 5' flanking region was deleted. SCHs an d chromosome 7-enriched YACs were used to demonstrate that all four hY RNA genes reside on human chromosome 7.