Structural And Functional Analysis Of 5' External Transcribed Spacer In Saccharomyces Cerevisiae Precursor RRNA

Eukaryotic ribosome biogenesis begins in the nucleolus with the transcription of a long precursor r RNA(pre-r RNA)that encodes the 5'-external transcribed spacer(5'ETS),18 s r RNA,internal transcribed spacer1(ITS1),5.8S r RNA,ITS2,25 S r RNA and 3'ETS in the 5' to 3' order.The four transcribed spacers are to be removed during ordered processing.The ribosomal RNA,18 S,5.8S and 25 S genome sequence tends to be conserved in most organisms,but 5'ETS,ITS1,ITS2 and 3'ETS as non-coding area,there are more changes.However,the 5' external transcribed spacer(5'ETS)is essential for the18 S r RNA processing.We analyzed the structure and function of 5'ETS of Saccharomyces cerevisiae.We have revised the secondary structure model of 5' ETS based on phylogenetic analysis of yeast sequences.By using a pre-r RNA expression plasmid system,we have undertaken a deletion and mutation analysis of the 5'ETS and examined production of 18 S r RNA.We constructed a series of pre-r RNA fragments fused to a MS2 and TOB RNA tag and expressed them from plasmid under the control of GAL7 promoter.We use plasmid complementary growth assay and Northern blot assay to analyze the functional sequences of 5'ETS.We affinity purified the in vivo assembled plasmid-derived pre-r RNA derived pre-r RNA complexes using the MS2 RNA tag and a TAP tagged protein as baits and identified the associated proteins with semi-quantitative mass spectrometry.According to our results,we conclude that there only a few part of the 5'ETS in Saccharomyces cerevisiae is essential and affect the 18 S r RNA processing.The sequence 5' of U3 binding site A is important.The linker between H2 and H3,the upper stem of H3 and H4,the remaining sequences are critical for 18 S processing.This region has been shown to recruit the UTPA complex and the key elements are likely directly recognized.5' region of H5 is required for a lot of 5'ETS factors recruitment.H6 is necessary for 5' ETS processing but there was no 90 S assembly factors absence without H6.Removal of the sequences nearby the A0 site and H10 also caused defective in 18 S processing.The sequences of H2 upper stem,H3 lower stem,H7,H10 and U3 binding site B are also important for 18 S processing,but deletion or mutation of these regions could not completely block the production of 18 S r RNA.By contrast,H1,H2 lower stem,H3 upper stem,H5 lower stem,H8,H9 are likely not required for the 18 S processing.We identified essential elements distributed along 5'ETS.The work reveals the structure and function of 5'ETS and provides insight into its function in 18 S processing.