ELEMENTS ESSENTIAL FOR PROCESSING INTRONIC U14 SNORNA ARE LOCATED AT THE TERMINI OF THE MATURE SNORNA SEQUENCE AND INCLUDE CONSERVED NUCLEOTIDE BOX-C AND BOX-D

Essential elements for intronic U14 processing have been analyzed by m icroinjecting various mutant hsc70/U14 pre-mRNA precursors into Xenopu s oocyte nuclei. Initial truncation experiments revealed that elements sufficient for U14 processing are located within the mature snoRNA se quence itself. Subsequent deletions within the U14 coding region demon strated that only the terminal regions of the folded U14 molecule cont aining conserved nucleotide boxes C and D are required for processing. Mutagenesis of either box C or box D completely blocked U14 processin g. The importance of boxes C and D was confirmed with the excision of appropriately sized U3 and U8 fragments containing boxes C and D from an hsc70 pre-mRNA intron. Competition studies indicate that a trans-ac ting factor (protein?) is binding this terminal motif and is essential for U14 processing. Competition studies also revealed that this facto r is common to both intronic and non-intronic snoRNAs possessing nucle otide boxes C and D. Immunoprecipitation of full-length and internally deleted U14 snoRNA molecules demonstrated that the terminal region co ntaining boxes C and D does not bind fibrillarin. Collectively, our re sults indicate that a trans-acting factor (different from fibrillarin) binds to the box C- and D-containing terminal motif of U14 snoRNA, th ereby stabilizing the intronic snoRNA sequence in an RNP complex durin g processing.