The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We ...therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using silica beads. In a variant approach (PAR‐TRAPP), RNAs were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR‐TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR‐TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA–peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization.
Synopsis
This study presents TRAPP (total RNA‐associated protein purification), a large‐scale approach that allows the rapid identification of all RNA‐binding proteins and quantification of dynamic changes following exposure to stress.
TRAPP allows rapid identification of the RNA‐bound proteome in both eukaryotes (S. cerevisiae) and bacteria (E. coli).
PAR‐TRAPP in yeast quantified changes in the RNA‐bound proteome on stress, revealing specific defects in 60S ribosome maturation.
iTRAPP mapped 524 unique RNA‐peptide crosslinks from 178 proteins, with amino acid resolution.
This study presents TRAPP (total RNA‐associated protein purification), a large‐scale approach that allows the rapid identification of all RNA‐binding proteins and quantification of dynamic changes following exposure to stress.
The 5′‐exonuclease Rat1 degrades pre‐rRNA spacer fragments and processes the 5′‐ends of the 5.8S and 25S rRNAs. UV crosslinking revealed multiple Rat1‐binding sites across the pre‐rRNA, consistent ...with its known functions. The major 5.8S 5′‐end is generated by Rat1 digestion of the internal transcribed spacer 1 (ITS1) spacer from cleavage site A3. Processing from A3 requires the ‘A3‐cluster’ proteins, including Cic1, Erb1, Nop7, Nop12 and Nop15, which show interdependent pre‐rRNA binding. Surprisingly, A3‐cluster factors were not crosslinked close to site A3, but bound sites around the 5.8S 3′‐ and 25S 5′‐regions, which are base paired in mature ribosomes, and in the ITS2 spacer that separates these rRNAs. In contrast, Nop4, a protein required for endonucleolytic cleavage in ITS1, binds the pre‐rRNA near the 5′‐end of 5.8S. ITS2 was reported to undergo structural remodelling. In vivo chemical probing indicates that A3‐cluster binding is required for this reorganization, potentially regulating the timing of processing. We predict that Nop4 and the A3 cluster establish long‐range interactions between the 5.8S and 25S rRNAs, which are subsequently maintained by ribosomal protein binding.
Ribosome biogenesis involves a number of pre‐rRNA cleavage events. This study characterizes the pre‐rRNA interaction sites of the ribosome synthesis factors Cic1, Erb1, Nop7, Nop12, Nop15 and Nop4 and the 5′‐exonuclease Rat1 and their role in 5.8S rRNA processing.
The U3 small nucleolar ribonucleoprotein (snoRNP) plays an essential role in ribosome biogenesis but, like many RNA-protein complexes, its architecture is poorly understood. To address this problem, ...binding sites for the snoRNP proteins Nop1, Nop56, Nop58, and Rrp9 were mapped by UV cross-linking and analysis of cDNAs. Cross-linked protein-RNA complexes were purified under highly-denaturing conditions, ensuring that only direct interactions were detected. Recovered RNA fragments were amplified after linker ligation and cDNA synthesis. Cross-linking was successfully performed either in vitro on purified complexes or in vivo in living cells. Cross-linking sites were precisely mapped either by Sanger sequencing of multiple cloned fragments or direct, high-throughput Solexa sequencing. Analysis of RNAs associated with the snoRNP proteins revealed remarkably high signal-to-noise ratios and identified specific binding sites for each of these proteins on the U3 RNA. The results were consistent with previous data, demonstrating the reliability of the method, but also provided insights into the architecture of the U3 snoRNP. The snoRNP proteins were also cross-linked to pre-rRNA fragments, with preferential association at known sites of box C/D snoRNA function. This finding demonstrates that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment. The techniques reported here should be widely applicable to analyses of RNA-protein interactions.
Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA ...maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.
The exosome complex of 3′–5′ exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro ...activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the
Trf4p/
Air2p/
Mtr4p
polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.
Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms ...by which two of the largest multi-protein complexes-UtpA and UtpB-interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA-protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5' end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5' ETS and U3 snoRNA as well as the 3' boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.
We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and ...coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.
We identified a complex in S. cerevisiae, the “exosome,” consisting of the five essential proteins Rrp4p, Rrp41p, Rrp42p, Rrp43p, and Rrp44p (Dis3p). Remarkably, four of these proteins are homologous ...to characterized bacterial 3′→5′ exoribonucleases; Rrp44p is homologous to RNase II, while Rrp41p, Rrp42p, and Rrp43p are related to RNase PH. Recombinant Rrp4p, Rrp44p, and Rrp41p are 3′→5′ exoribonucleases in vitro that have distributive, processive, and phosphorolytic activities, respectively. All components of the exosome are required for 3′ processing of the 5.8S rRNA. Human Rrp4p is found in a comparably sized complex, and expression of the
hRRP4 gene in yeast complements the
rrp4-1 mutation. We conclude that the exosome constitutes a highly conserved eukaryotic RNA processing complex.
In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in ...extramitochondrial iron–sulfur (Fe–S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe–S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe–S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre‐rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre‐40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe–S proteins.
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