Ribosomes are RNA-based molecular machines for protein synthesis that are produced in a cell in a complex assembly pathway (1
). The main structural components of the eukaryotic ribosome are 18S rRNA in the small subunit and 5.8S/25S rRNAs (5.8S/28S in higher eukaryotes) in the large subunit. These rRNAs are initially transcribed by RNA polymerase I (Pol I) as a single large precursor, which also contains spacer regions removed during pre-rRNA maturation. For example, the primary transcript in mammalian species, 47S pre-rRNA, contains external (5′ETS, 3′ETS) and internal (ITS1 and ITS2) transcribed spacers (A, Supplementary Figure S1
). The synthesis of ribosomal subunits involves several endonucleolytic cleavages within spacer regions followed by exonucleolytic trimming to form the 5′ and 3′ ends of the mature rRNAs (2
). In Saccharomyces cerevisiae
, 5′ exonucleases function in the maturation of the 5′ ends of 5.8S and 25S rRNAs (4
). In addition, several 3′ exonucleases have been implicated in the generation of the 3′ end of 5.8S rRNA (6–11
). Why these rRNA ends are generated through exonuclease trimming rather than precise cleavages is not exactly clear.
Figure 1. Xrn2 contributes to the degradation of incomplete Pol I transcripts. (A) Structure of the full-length mouse Pol I transcript (47S pre-rRNA) showing major processing sites. (B) Incubation of cells with low doses (20ng/ml) of ActD leads to abortive (more ...)
The complex nature of ribosome assembly leads to inevitable errors in processing and folding of pre-rRNA. Surveillance mechanisms are crucial for eliminating defective precursors, thereby promoting structural and functional integrity of the final ribosomal particles (12
). The exosome, a multisubunit protein complex containing 3′ exoribonuclease activities (6
), plays an important role in rRNA quality control by initiating 3′ to 5′ degradation of defective pre-rRNAs in yeast (13
). In many cases, it is still not well understood how the correct and defective rRNA precursors can be distinguished by the surveillance machinery. The TRAMP (Trf4/5p-Air1/2p-Mtr4p polyadenylation) complex is thought to provide an important regulatory role by tagging degradation substrates with short poly(A) tails that facilitate degradation by the exosome (15–17
Ribosome biogenesis in mammals has been much less studied than in yeast, although many factors involved in this pathway are predicted to perform similar functions across eukaryotic species. Polyadenylated degradation intermediates have been identified among different RNA classes including rRNA in mammalian cells, suggesting conservation of the TRAMP/exosome surveillance function (18
). We previously showed that aberrant Pol I transcripts in mouse cells can undergo polyadenylation, which is dependent on the mouse Trf4p homolog Papd5, and that degradation of these polyadenylated species involves the core exosome and an associated nuclear 3′ exonuclease, Exosc10 (PM/Scl-100) (19
). The mammalian exosome and Exosc10 are present in the nucleolus (20
), and are expected to be functionally analogous to their yeast counterparts with regard to pre-rRNA processing, although this remains to be experimentally demonstrated. The role of 5′ exonucleases in pre-rRNA maturation in mammalian species, to the best of our knowledge, has not been previously evaluated.
In this study, we report that both processing and decay of pre-rRNAs in mammalian cells rely heavily upon the 5′ to 3′ degradation carried out by the nuclear 5′ exonuclease Xrn2. A systematic evaluation of the mouse pre-rRNA maturation pathway revealed that multiple 5′ ends generated by endonucleolytic cleavages in pre-rRNA become targets for Xrn2. The mature 5′ ends of 5.8S and 28S rRNAs in mouse cells are processed by Xrn2 from longer 5′-extended precursors, demonstrating for the first time the conservation of an exonucleolytic trimming mechanism in their formation from yeast to mammals. Unexpectedly, abortive Pol I transcripts, discarded spacer fragments and aberrant precursors are all actively degraded from 5′ to 3′ and accumulate in cells upon Xrn2 depletion. Our data support a model in which proofreading of 5′ ends by Xrn2 is used repeatedly during pre-rRNA maturation as a mechanism to determine whether pre-rRNA intermediates are to be processed to mature forms or discarded.