ribosomal protein gene

The importance of introns for the expression of the mouse ribosomal protein (rp) gene rpL32 was evaluated by transfection experiments with various mutant constructs. We found that introns have both a general and a specific role in rpL32 expression. The general role can be satisfied by any spliceable intron, while the specific role is uniquely attributable to intron 1, which contains a transcriptional regulatory element. Using gel-mobility-shift assays, methylation interference analysis, and site-specific mutagenesis, the intronic transcriptional element was defined as the sequence motif 5$\sp\prime$-GGCGGCCATC-3$\sp\prime$ (+60 to +69), which interacts with a specific nuclear factor $\delta$ . This element was inactive when translocated to a position upstream of the transcriptional start-site or to a position within the third intron, indicating that it does not have the properties of a typical enhancer. Additional experiments indicated that these intronic elements functions independently of an exonic $\delta$ factor-binding element that is located 33 base pairs upstream. Concerning the general role of a spliceable intron, our results demonstrated that it does not affect the efficiency of transcriptional initiation. Rather, association with spliceosomes seems to stabilize primary transcripts in the nucleus and to be required for efficient export of mRNA from nucleus to cytoplasm. Although attempts to transcribe the rpL32 gene in vitro using either a whole cell extract or a high-efficiency nuclear extract prepared from HeLa cells were unsuccessful, these systems were able to accurately transcribe another mouse rp gene, rpS16, and to recognize all the rpS16 promoter elements that were functionally identified in vivo. The importance of a promoter proximal element located at the conventional TATA box region and an initiator element, which spans the transcriptional start-site, were assessed using an appropriate set of mutants. Consistent with previous in vivo results, the initiator was found to be the primary determinant for initiation-site selection. However, some discrepancies between the in vitro and in vivo results were also observed, indicating that the in vitro system is biased towards sequence elements that are not present in the natural rpS16 gene