Cis-acting elements in the 5' end of the internal transcribed spacer 1 from Schizosaccharomyces pombe pre-rRNA
Internal transcribed spacers are critical for rRNA processing and maturation. They work as organizers of a variety of protein factors that initially bind to the extended hairpin in the spacers. This primary processing complex appears to target the smaller hairpins in the spacer, and may serve as a substrate for nucleases to start the cleavage process. The present research was focused on learning more about the role of the 5' end of ITS 1 during rRNA maturation, both with respect to sequence and secondary structure. A previously developed experimental system consisting of a cloned rDNA transcription unit and two neutral tags in the 5.8S and 18S rRNA sequences, was used to examine mutations ' in vivo'. Different substitutions were introduced in the 5' end of ITS 1 and their effects on 5.8S and 18S rRNA were examined. Although the region was not found to be indispensable for RNA processing and ribosome maturation, it was found to contribute to a normal and efficient process. As a further step to delineate any critical elements in the 3'end of 18S and 5'end of ITS 1, preliminary experiments were undertaken to better define the intermediate termini and related cleavage sites. Preliminary results using RNA ligation and PCR amplification followed with DNA sequencing suggest that the approach could provide good data with further implementation. Taken together all the results provide further evidence that the rRNA processing pathway is highly interconnected and dependant on a large number of trans and cis-acting factors where the spacers act as "biological springs", that help to organize and bring together the different domains that need to be cut.