Abstract
In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.
Highlights
The biogenesis of eukaryotic ribosomes is a massive undertaking involving binding of ribosomal proteins (r-proteins) to ribosomal RNA (rRNA) and processing of precursor rRNA along pathways that are facilitated by over 250 ribosomal assembly factors [1,2]
The precursor transcript associates with a large number of ribosomal assembly factors to form the “90S” precursor particle, which is subsequently split into subunit-specific entities by cutting the precursor rRNA at the A2 site in ITS1 (Figure 1A)
Electron micrographs of yeast rRNA genes associated with RNA transcripts show that rRNA for both the small and large subunits is folded during transcription [6]
Summary
The biogenesis of eukaryotic ribosomes is a massive undertaking involving binding of ribosomal proteins (r-proteins) to rRNA and processing of precursor rRNA along pathways that are facilitated by over 250 ribosomal assembly factors [1,2]. The External Transcribed Spacers (5 and 3 ETS) make up the 5 and 3 parts of the primary transcript, while the Internal Transcribed Spacers (ITS1 and ITS2) are interstitial between the 18S, 5.8S, and 25S rRNA parts. As it is transcribed, the precursor transcript associates with a large number of ribosomal assembly factors to form the “90S” precursor particle, which is subsequently split into subunit-specific entities by cutting the precursor rRNA at the A2 site in ITS1 (Figure 1A). J.tMheol.tSrcai.n2s0c1r9i,p20t,iox nFOaRnPdEEpRroRcEeVsIEsWing of the rRNA and chaperoned to the nucleolus o2r nofu1c5leus where they associate with the nascent ribosomal subunits in a hierarchical fashion [7,8,9]
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