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The RNA exosome, a major RNA degradation machine, processes ribosomal RNA (rRNA) precursors and is directly coupled to the protein synthesis machine, the ribosome. Using cryo–electron microscopy, Schuller et al. investigated the structure of the precursor large ribosomal subunit from yeast with unprocessed rRNA in complex with the RNA exosome. The structure captures a snapshot of two molecular machines transiently interacting and explains how the RNA exosome acts on an authentic physiological substrate and remodels the large subunit during ribosome maturation. Science , this issue p. 219 The exosome forms a single structural and functional unit with its preribosomal substrate to catalyze ribosomal RNA processing and ribosome maturation. The RNA exosome complex processes and degrades a wide range of transcripts, including ribosomal RNAs (rRNAs). We used cryo–electron microscopy to visualize the yeast nuclear exosome holocomplex captured on a precursor large ribosomal subunit (pre-60 S ) during 7 S -to-5.8 S rRNA processing. The cofactors of the nuclear exosome are sandwiched between the ribonuclease core complex (Exo-10) and the remodeled “foot” structure of the pre-60 S particle, which harbors the 5.8 S rRNA precursor. The exosome-associated helicase Mtr4 recognizes the preribosomal substrate by docking to specific sites on the 25 S rRNA, captures the 3′ extension of the 5.8 S rRNA, and channels it toward Exo-10. The structure elucidates how the exosome forms a structural and functional unit together with its massive pre-60 S substrate to process rRNA during ribosome maturation.

We developed a novel technique, called pseudouridine site identification sequencing (PSI-seq), for the transcriptome-wide mapping of pseudouridylation sites with single-base resolution from cellular RNAs based on the induced termination of reverse transcription specifically at pseudouridines following CMCT treatment. PSI-seq analysis of RNA samples from S. cerevisiae correctly detected all of the 43 known pseudouridines in yeast 18S and 25S ribosomal RNA with high specificity. Moreover, application of PSI-seq to the yeast transcriptome revealed the presence of site-specific pseudouridylation within dozens of mRNAs, including RPL11a, TEF1, and other genes implicated in translation. To identify the mechanisms responsible for mRNA pseudouridylation, we genetically deleted candidate pseudouridine synthase (Pus) enzymes and reconstituted their activities in vitro. These experiments demonstrated that the Pus1 enzyme was necessary and sufficient for pseudouridylation of RPL11a mRNA, whereas Pus4 modified TEF1 mRNA, and Pus6 pseudouridylated KAR2 mRNA. Finally, we determined that modification of RPL11a at Ψ -68 was observed in RNA from the related yeast S. mikitae, and Ψ -239 in TEF1 mRNA was maintained in S. mikitae as well as S. pombe, indicating that these pseudouridylations are ancient, evolutionarily conserved RNA modifications. This work establishes that site-specific pseudouridylation of eukaryotic mRNAs is a genetically programmed RNA modification that naturally occurs in multiple yeast transcripts via distinct mechanisms, suggesting that mRNA pseudouridylation may provide an important novel regulatory function. The approach and strategies that we report here should be generally applicable to the discovery of pseudouridylation, or other RNA modifications, in diverse biological contexts.

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. This complicated processing reaction begins with a single endonucleolytic cleavage followed by exonucleolytic trimming at both new cleavage sites to generate mature 5.8S and 25S rRNA. We reconstituted the 7S→5.8S processing branch within ITS2 using purified exosome and its nuclear cofactors. We find that both Rrp44’s ribonuclease activities are required for initial RNA shortening followed by hand over to the exonuclease Rrp6. During the in vitro reaction, ITS2-associated factors dissociate and the underlying ‘foot’ structure of the pre-60S particle is dismantled. 7S pre-rRNA processing is independent of 5S RNP rotation, but 26S→25S trimming is a precondition for subsequent 7S→5.8S processing. To complete the in vitro assay, we reconstituted the entire cycle of ITS2 removal with a total of 18 purified factors, catalysed by the integrated activities of the two participating RNA-processing machines, the Las1 complex and nuclear exosome. Excision of internal transcribed spacer 2 (ITS2) within eukaryotic pre-ribosomal RNA is essential for ribosome function. Here, the authors reconstitute the entire cycle of ITS2 processing in vitro using purified components, providing insights into the cleavage process and demonstrating that 26S pre-rRNA processing necessarily precedes 7S pre-rRNA processing.

Modified nucleotide 5-methylcytosine (m5C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA:m5C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m5C- MTase activity, which restores m5C formation at position 2870 in domain V of 25S rRNA in a nop2Δ yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m5C in domains V and IV, respectively. In addition, we do not find any evidence of m5C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m5C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m5C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m5C:RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes.

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit. The molecular events underlying the assembly and maturation of the early pre-60S particles during eukaryotic ribosome synthesis are not well understood. Here, the authors combine yeast genetics and biochemical experiments to characterise the functions of two important players of eukaryotic ribosome biogenesis, the box C/D snoRNP snR190 and the helicase Dbp7, which both interact. They show that the snR190 snoRNA acts as a RNA chaperone that assists the structuring of the 25S rRNA during the maturation of early pre-60S particles and that Dbp7 is important for facilitating remodeling events in the peptidyl transferase center region of the 25S rRNAs during the maturation of early pre-60S particles.

Ribosome biogenesis is an essential and tightly regulated process linked to cell proliferation and growth. However, its regulatory mechanisms in oomycetes, a group of organisms with significant agricultural and ecological importance, remain unclear. In this study, we identify Puf4, a non-classical PUF (Pumilio and FBF) family RNA-binding protein that plays a conserved and crucial role in pre-rRNA processing in oomycetes. Knockout of PuPuf4 in Pythium ultimum or its ortholog PsPuf4 in Phytophthora sojae results in defective vegetative growth, impaired development, and reduced pathogenicity. Specifically, PuPuf4 binds to the H68 component of 25S rRNA, and its knockout leads to overaccumulation of rRNA processing intermediates, including 5′ETS, ITS1, and ITS2 precursors. Additionally, the AG-rich motif identified as the first binding motif of L-shaped PUF proteins, including PuPuf4, APUM24, and ScPuf6, may contribute to their specific RNA-binding affinity due to its unique structural features. Given the conserved role of Puf4 in oomycete pathogenicity, we developed the first nano-material-free dsRNA delivery system via zoospore-specific uptake, effectively attenuating virulence in Pythium aphanidermatum and Ph. sojae through RNAi targeting Puf4 . This study presents novel findings on structural and functional conservation of Puf4 and offers a promising RNAi-based strategy for controlling oomycete plant diseases.

Small nucleolar RNAs are non-coding transcripts that guide chemical modifications of RNA substrates and modulate gene expression at the epigenetic and post-transcriptional levels. However, the extent of their regulatory potential and the underlying molecular mechanisms remain poorly understood. Here, we identify a conserved, previously unannotated intronic C/D-box snoRNA, termed snR107 , hosted in the fission yeast long non-coding RNA mamRNA and carrying two independent cellular functions. On the one hand, snR107 guides site-specific 25S rRNA 2’-O-methylation and promotes pre-rRNA processing and 60S subunit biogenesis. On the other hand, snR107 associates with the gametogenic RNA-binding proteins Mmi1 and Mei2, mediating their reciprocal inhibition and restricting meiotic gene expression during sexual differentiation. Both functions require distinct cis -motifs within snR107 , including a conserved 2’-O-methylation guiding sequence. Together, our results position snR107 as a dual regulator of rRNA modification and gametogenesis effectors, expanding our vision on the non-canonical functions exerted by snoRNAs in cell fate decisions. Small nucleolar RNAs (snoRNAs) are known for their role in RNA modification to regulate gene expression. Here, the authors identify a snoRNA that not only guides ribosomal RNA 2’- O-methylation but also modulates the activities of RNA-binding proteins involved in fission yeast gametogenesis.

Plant ribosome-inactivating protein (RIP) toxins are EC3.2.2.22 N-glycosidases, found among most plant species encoded as small gene families, distributed in several tissues being endowed with defensive functions against fungal or viral infections. The two main plant RIP classes include type I (monomeric) and type II (dimeric) as the prototype ricin holotoxin from Ricinus communis that is composed of a catalytic active A chain linked via a disulphide bridge to a B-lectin domain that mediates efficient endocytosis in eukaryotic cells. Plant RIPs can recognize a universally conserved stem-loop, known as the α-sarcin/ ricin loop or SRL structure in 23S/25S/28S rRNA. By depurinating a single adenine (A4324 in 28S rat rRNA), they can irreversibly arrest protein translation and trigger cell death in the intoxicated mammalian cell. Besides their useful application as potential weapons against infected/tumor cells, ricin was also used in bio-terroristic attacks and, as such, constitutes a major concern. In this review, we aim to summarize past studies and more recent progresses made studying plant RIPs and discuss successful approaches that might help overcoming some of the bottlenecks encountered during the development of their biomedical applications.