Assembly Map Of The Earliest Precursors Of Large Ribosomal Subunits In Yeast

Ribosome,the organalle that synthesizes proteins,is a large RNA-protein complex composed of a small and large subunit.In the yeast Saccharomyces cerevisiae,the small subunit is composed of18S rRNA and 33 ribosomal proteins and the large subunit is composed of 25S,5.8S and 5S rRNAs and 46 ribosomal proteins.Ribosome synthesis is a fundamental and complicated undertaking of cells.Ribosome biogenesis begins in the nucleolus with the transcription of a 35S precursor rRNA(pre-rRNA).35S pre-rRNA encodes the 5' external transcribed spacer(5'ETS),18S rRNA,internal transcribed spacer 1(ITS1),5.8S rRNA,internal transcribed spacer 2(ITS2),25S rRNA and 3' external transcribed spacer(3'ETS).The 5' half of 35S pre-rRNA is assembled with many ribosomal proteins,snoRNAs and numerous non-ribosomal factors to form the 90S preribosome.After processing and modification of pre-rRNA,90S is converted into a pre-40S particle that eventually develops into the 40S small subunit.The 3' half of 35S pre-rRNA(called pre-27S)is assembled into pre-60S particles,which undergo complicated maturation steps before forming the mature 60S large subunits.In yeast,the assembly of ribosome occurs cotranscriptionally.The assembly factors and ribosomal proteins combine with the nascent pre-rRNA transcript,forming the earliest precursor ribosome particles.The cotranscriptional assembly of 90S particle and pre-40S particle have been studied in details.However,the cotranscriptional assembly process of pre-60S particle is not clearly understood,since the cotranscriptional assembly of pre-60S occurs rapidly and such pre-60S particles are difficult to purify and analyze.The mature 25 S rRNA is composed of six domains that are highly interwined in the 60S structure,perhaps the earliest pre-60S particles are assembled on the full-length pre-rRNA in a cooperative manner.To study the cotranscriptional assembly process of pre-60S particle,I constructed a series of plasmids driven by a GAL promoter to express the pre-27S fragments with increasing lengths.These RNAs end at each of six domains of 25S rRNA to mimic the nascent pre-27S transcripts.I affinity purified the in vivo assembled pre-60S particles via a MS2 RNA tag inserted at the ITS2 and the TAP-tagged Nop7 protein,which is an early assembly factor in pre-60S.The purified particles were analyzed by mass spectrometry to identify the associated proteins and by northern blot to identify the processing status of pre-rRNA and 5S rRNA.Our study reveals a spatiotemporal stepwise assembly map for 34 assembly factors,30 ribosomal proteins and 5S rRNA.The gradual association of assembly factors and ribosomal proteins with the pre-27S fragments strongly supports that the pre-60S is co-transcriptionally,rather than post-transcriptionally,assembled.The assembly map also reveals the approximate binding sites of these assembly factors.The 5' half of pre-27S primarily recruited A3 factors required for ITS1 processing and the 3' half of pre-27S recruited most B factors required for ITS2 processing.This thesis shows that processing of both ITS1 and ITS2 requires the entire 25S rRNA to be transcribed,5S rRNA is assembled upon completion of 25S.The 3'ETS plays a minor role in ribosome assembly,but is important for efficient rRNA processing and ribosome maturation.The particle purified with the bait protein Ssfl was previously considered to be the earliest pre-60S particle.However,the plasmid-derived fully-assembled pre-60S particles contain an uncleaved ITS2 region and a simpler composition of proteins and represent a more primitive state than the Ssfl particle.Overall,this work elucidates the elusive co-transcriptional assembly process of large ribosomal subunit.