RESUMO
Ribosome synthesis is an essential cellular process, and perturbation of human ribosome production is linked to cancer and genetic diseases termed ribosomopathies. During their assembly, pre-ribosomal particles undergo numerous structural rearrangements, which establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome biogenesis. RNA helicases are essential mediators of such remodelling events and here, we demonstrate that the DEAH-box RNA helicase DHX37 is required for maturation of the small ribosomal subunit in human cells. Our data reveal that the presence of DHX37 in early pre-ribosomal particles is monitored by a quality control pathway and that failure to recruit DHX37 leads to pre-rRNA degradation. Using an in vivo crosslinking approach, we show that DHX37 binds directly to the U3 small nucleolar RNA (snoRNA) and demonstrate that the catalytic activity of the helicase is required for dissociation of the U3 snoRNA from pre-ribosomal complexes. This is an important event during ribosome assembly as it enables formation of the central pseudoknot structure of the small ribosomal subunit. We identify UTP14A as a direct interaction partner of DHX37 and our data suggest that UTP14A can act as a cofactor that stimulates the activity of the helicase in the context of U3 snoRNA release.
Assuntos
RNA Helicases DEAD-box/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Ribossomos/metabolismo , Catálise , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Nucleolar Pequeno/genéticaRESUMO
Production of eukaryotic ribosomal subunits is a highly dynamic process; pre-ribosomes undergo numerous structural rearrangements that establish the architecture present in mature complexes and serve as key checkpoints, ensuring the fidelity of ribosome assembly. Using in vivo crosslinking, we here identify the pre-ribosomal binding sites of three RNA helicases. Our data support roles for Has1 in triggering release of the U14 snoRNP, a critical event during early 40S maturation, and in driving assembly of domain I of pre-60S complexes. Binding of Mak5 to domain II of pre-60S complexes promotes recruitment of the ribosomal protein Rpl10, which is necessary for subunit joining and ribosome function. Spb4 binds to a molecular hinge at the base of ES27 facilitating binding of the export factor Arx1, thereby promoting pre-60S export competence. Our data provide important insights into the driving forces behind key structural remodelling events during ribosomal subunit assembly.
Assuntos
RNA Helicases DEAD-box/metabolismo , Subunidades Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiaeRESUMO
Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Herein, we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N6 -methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. The developed deoxyribozymes are sensitive to the presence of N6 -methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m6 A as a regulatory element that influences RNA folding and protein binding.
Assuntos
Adenosina/análogos & derivados , DNA Catalítico/metabolismo , RNA/metabolismo , Adenosina/análise , Adenosina/metabolismo , Sequência de Bases , Metilação , Conformação de Ácido Nucleico , RNA/química , Clivagem do RNA , Especificidade por SubstratoRESUMO
N6-methyladenosine (m6A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m6As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed "readers" that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m6A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m6A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5'-3' exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a "fast-track" expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation.
Assuntos
Adenosina Trifosfatases/metabolismo , Adenosina/análogos & derivados , Exorribonucleases/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Adenosina/química , Adenosina/metabolismo , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/química , RNA/genética , RNA/metabolismo , RNA Helicases , Relação Estrutura-AtividadeRESUMO
Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2'-O-methylated nucleotides, which are largely introduced by small Cajal body-specific ribonucleoproteins (scaRNPs). Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNA splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.
Assuntos
RNA Nuclear Pequeno/metabolismo , Spliceossomos/metabolismo , HumanosRESUMO
RNA helicases are critical regulators at the nexus of multiple pathways of RNA metabolism, and in the complex cellular environment, tight spatial and temporal regulation of their activity is essential. Dedicated protein cofactors play key roles in recruiting helicases to specific substrates and modulating their catalytic activity. Alongside individual RNA helicase cofactors, networks of cofactors containing evolutionarily conserved domains such as the G-patch and MIF4G domains highlight the potential for cross-regulation of different aspects of gene expression. Structural analyses of RNA helicase-cofactor complexes now provide insight into the diverse mechanisms by which cofactors can elicit specific and coordinated regulation of RNA helicase action. Furthermore, post-translational modifications (PTMs) and long non-coding RNA (lncRNA) regulators have recently emerged as novel modes of RNA helicase regulation.
Assuntos
Biocatálise , Processamento de Proteína Pós-Traducional , RNA Helicases/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Ativação Enzimática , Humanos , RNA Helicases/químicaRESUMO
Mitochondrial protein synthesis is essential for the production of components of the oxidative phosphorylation system. RNA modifications in the mammalian mitochondrial translation apparatus play key roles in facilitating mitochondrial gene expression as they enable decoding of the non-conventional genetic code by a minimal set of tRNAs, and efficient and accurate protein synthesis by the mitoribosome. Intriguingly, recent transcriptome-wide analyses have also revealed modifications in mitochondrial mRNAs, suggesting that the concept of dynamic regulation of gene expression by the modified RNAs (the "epitranscriptome") extends to mitochondria. Furthermore, it has emerged that defects in RNA modification, arising from either mt-DNA mutations or mutations in nuclear-encoded mitochondrial modification enzymes, underlie multiple mitochondrial diseases. Concomitant advances in the identification of the mitochondrial RNA modification machinery and recent structural views of the mitochondrial translation apparatus now allow the molecular basis of such mitochondrial diseases to be understood on a mechanistic level.
Assuntos
Mitocôndrias/genética , RNA Mensageiro/genética , RNA de Transferência/genética , RNA/genética , Animais , Sequência de Bases , Humanos , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA de Transferência/química , RNA de Transferência/metabolismoRESUMO
N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A "writer" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.
Assuntos
Adenosina/análogos & derivados , Metiltransferases/genética , Precursores de RNA/genética , Splicing de RNA , RNA Longo não Codificante/metabolismo , RNA Nuclear Pequeno/metabolismo , Adenosina/metabolismo , Pareamento de Bases , Sequência de Bases , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Metilação , Metiltransferases/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Precursores de RNA/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
RNA methyltransferases (MTases) are responsible for co- and posttranscriptional methylation of nucleotides in a wide variety of RNA substrates. Examination of the target specificity, catalytic activity, and function of these enzymes requires in vitro methylation assays. Here, we provide a detailed protocol for the methylation of in vitro transcripts, synthetic RNAs, and total cellular RNA using recombinant RNA methyltransferases and S-adenosylmethionine (SAM) as a methyl group donor. We describe how this method can be coupled to fluorographic detection of RNA methylation if 3H-labeled SAM is used, and discuss alternative chromatography-based methods for the detection of methylated nucleotides, focusing on reversed-phase high-performance liquid chromatography (RP-HPLC). In both cases, mutagenesis of the methyltransferase or the RNA substrate can be employed to elucidate the catalytic mechanisms and target specificity of the enzymes. Together these approaches provide valuable insight into the action of RNA methyltransferases on the molecular level and serve as a basis for further functional characterization of RNA methyltransferases in vivo.
Assuntos
Ensaios Enzimáticos/métodos , Metiltransferases/metabolismo , RNA/genética , RNA/metabolismo , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Epigênese Genética , Epigenômica/métodos , Técnicas In Vitro , Metilação , RNA/química , Especificidade por SubstratoRESUMO
Several crosslinking methods have been developed to identify interacting RNAs for proteins of interest. Here, we describe variants of the UV crosslinking and analysis of cDNA (CRAC) method that allow target identification of RNA methyltransferases on a genome-wide scale. We present a detailed protocol for the application of CRAC in human cells that stably express the protein of interest fused to a tandem affinity tag. After the introduction of a covalent link between the protein and its target RNAs, protein-RNA complexes are purified and bound RNAs trimmed, ligated to adapters, reverse transcribed, and amplified. Sequences obtained from next-generation sequencing are then mapped onto the human genome allowing the identification of possible substrates. For some RNA methyltransferases, e.g., m5C MTases, their catalytic mechanism can be exploited for chemical crosslinking approaches instead of UV based crosslinking.
Assuntos
Metiltransferases/metabolismo , RNA/genética , RNA/metabolismo , Azacitidina/química , Azacitidina/metabolismo , Cromatografia de Afinidade , Biologia Computacional/métodos , Citidina/análogos & derivados , Citidina/química , Citidina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Metiltransferases/isolamento & purificação , RNA/química , Estatística como Assunto/métodos , Especificidade por Substrato , Raios UltravioletaRESUMO
Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA-protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3Î half of the snR30 snoRNA. Protein-protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation.
Assuntos
Proteínas Nucleares/biossíntese , Conformação de Ácido Nucleico , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos/genética , Mapas de Interação de Proteínas/genética , Precursores de RNA/genética , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Nucleolar Pequeno/biossíntese , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribossomos/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In eukaryotes, the synthesis of ribosomal subunits, which involves the maturation of the ribosomal (r)RNAs and assembly of ribosomal proteins, requires the co-ordinated action of a plethora of ribosome biogenesis factors. Many of these cofactors remain to be characterized in human cells. Here, we demonstrate that the human G-patch protein NF-κB-repressing factor (NKRF) forms a pre-ribosomal subcomplex with the DEAH-box RNA helicase DHX15 and the 5Î-3Î exonuclease XRN2. Using UV crosslinking and analysis of cDNA (CRAC), we reveal that NKRF binds to the transcribed spacer regions of the pre-rRNA transcript. Consistent with this, we find that depletion of NKRF, XRN2 or DHX15 impairs an early pre-rRNA cleavage step (A'). The catalytic activity of DHX15, which we demonstrate is stimulated by NKRF functioning as a cofactor, is required for efficient A' cleavage, suggesting that a structural remodelling event may facilitate processing at this site. In addition, we show that depletion of NKRF or XRN2 also leads to the accumulation of excised pre-rRNA spacer fragments and that NKRF is essential for recruitment of the exonuclease to nucleolar pre-ribosomal complexes. Our findings therefore reveal a novel pre-ribosomal subcomplex that plays distinct roles in the processing of pre-rRNAs and the turnover of excised spacer fragments.
Assuntos
Exorribonucleases/metabolismo , Biogênese de Organelas , RNA Helicases/metabolismo , Proteínas Repressoras/metabolismo , Ribossomos/metabolismo , Biocatálise , Nucléolo Celular/metabolismo , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Ligação Proteica , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Subunidades Ribossômicas/metabolismoRESUMO
The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.
Assuntos
RNA Helicases DEAD-box/genética , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Bromovirus/genética , Éxons/genética , Regulação da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , Ribossomos/genética , Saccharomyces cerevisiae/genéticaRESUMO
rRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.
Assuntos
Células Eucarióticas/fisiologia , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Acetilação , Animais , Suscetibilidade a Doenças , Humanos , Metilação , RNA Ribossômico/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribossomos/química , Relação Estrutura-AtividadeRESUMO
Bowen-Conradi syndrome (BCS) is a severe genetic disorder that is characterised by various developmental abnormalities, bone marrow failure and early infant death. This disease is caused by a single mutation leading to the aspartate 86 to glycine (D86G) exchange in the essential nucleolar RNA methyltransferase EMG1. EMG1 is required for the synthesis of the small ribosomal subunit and is involved in modification of the 18S ribosomal RNA. Here, we identify the pre-ribosomal factors NOP14, NOC4L and UTP14A as members of a nucleolar subcomplex that contains EMG1 and is required for its recruitment to nucleoli. The BCS mutation in EMG1 leads to reduced nucleolar localisation, accumulation of EMG1D86G in nuclear foci and its proteasome-dependent degradation. We further show that EMG1 can be imported into the nucleus by the importins (Imp) Impα/ß or Impß/7. Interestingly, in addition to its role in nuclear import, binding of the Impß/7 heterodimer can prevent unspecific aggregation of both EMG1 and EMG1D86G on RNAs in vitro, indicating that the importins act as chaperones by binding to basic regions of the RNA methyltransferase. Our findings further indicate that in BCS, nuclear disassembly of the import complex and release of EMG1D86G lead to its nuclear aggregation and degradation, resulting in the reduced nucleolar recruitment of the RNA methyltransferase and defects in the biogenesis of the small ribosomal subunit.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Retardo do Crescimento Fetal/genética , Metiltransferases/genética , Proteínas Nucleares/genética , Transtornos Psicomotores/genética , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Retardo do Crescimento Fetal/patologia , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Transtornos Psicomotores/patologia , RNA Ribossômico 18S/genética , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Mitochondrial gene expression uses a non-universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt-)tRNAMet mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt-tRNAMet to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt-tRNAMet to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt-tRNAMet in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt-tRNAMet function. Together, our data reveal how modifications in mt-tRNAMet are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAMet to recognise the different codons encoding methionine.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Códon/metabolismo , Proteínas de Membrana/metabolismo , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , Animais , Humanos , Mamíferos , Análise de Sequência de DNARESUMO
During ribosomal RNA (rRNA) maturation, cleavages at defined sites separate the mature rRNAs from spacer regions, but the identities of several enzymes required for 18S rRNA release remain unknown. PilT N-terminus (PIN) domain proteins are frequently endonucleases and the PIN domain protein Utp24 is essential for early cleavages at three pre-rRNA sites in yeast (A0, A1 and A2) and humans (A0, 1 and 2a). In yeast, A1 is cleaved prior to A2 and both cleavages require base-pairing by the U3 snoRNA to the central pseudoknot elements of the 18S rRNA. We found that yeast Utp24 UV-crosslinked in vivo to U3 and the pseudoknot, placing Utp24 close to cleavage at site A1. Yeast and human Utp24 proteins exhibited in vitro endonuclease activity on an RNA substrate containing yeast site A2. Moreover, an intact PIN domain in human UTP24 was required for accurate cleavages at sites 1 and 2a in vivo, whereas mutation of another potential site 2a endonuclease, RCL1, did not affect 18S production. We propose that Utp24 cleaves sites A1/1 and A2/2a in yeast and human cells.