RESUMO
MicroRNAs (miRNAs) comprise a large family of regulatory molecules that repress protein production from targeted mRNAs. Although it is now clear that miRNAs exert pervasive effects on gene expression in animal cells, the mechanism(s) by which they function remains poorly understood. We have analyzed the subcellular distribution of miRNAs in actively growing HeLa cells and find that the vast majority are associated with actively translating mRNAs in polysomes. We also find that a specific miRNA-regulated mRNA (KRAS) is polysome associated and that its translation is impaired, apparently at the level of elongation. These observations are discussed in light of our current understanding of mechanism of miRNA function.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células HeLa , Humanos , Polirribossomos/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismoRESUMO
Trans-splicing requires that 5' and 3' splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3' splice site recognition in vitro. Efficient recognition of the 3' splice site is dependent upon both the sequence of the 3' splice site itself and enhancer elements located in the 3' exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.
Assuntos
Elementos Facilitadores Genéticos , Nematoides/genética , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Animais , Éxons , Mutação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genéticaRESUMO
A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.
Assuntos
Trifosfato de Adenosina/metabolismo , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Sistema Livre de Células , Células HeLa , Humanos , Dados de Sequência Molecular , Nematoides , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
This report describes a method that combines nuclease protection and site-specific labeling to determine sites and extents of RNA-protein interactions. The utility of the method is demonstrated by the analysis of the binding of factors to the 3' splice site region of a pre-mRNA labeled at three specific positions. This "reverse footprinting" technique should be widely applicable to a variety of questions concerning RNA-protein interactions.
Assuntos
Marcação por Isótopo/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Sítios de Ligação , Hidrólise , Ligação Proteica , RNA/análise , RNA/química , Precursores de RNA/análise , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/metabolismoRESUMO
Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.
Assuntos
Ascaris lumbricoides/genética , Splicing de RNA , RNA de Helmintos/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Animais , Ascaris lumbricoides/metabolismo , Autoantígenos/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Helmintos/química , RNA Mensageiro/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Proteínas Centrais de snRNPRESUMO
The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.
Assuntos
Ascaris/genética , Splicing de RNA , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Oxigênio/metabolismo , RNA de Helmintos/química , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.
Assuntos
Ascaris lumbricoides/genética , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/genética , Animais , Ascaris lumbricoides/embriologia , Sistema Livre de Células , Genes de Helmintos , Hibridização de Ácido Nucleico , Capuzes de RNA , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismoRESUMO
U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.
Assuntos
Ascaris lumbricoides/genética , Núcleo Celular/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Animais , Ascaris lumbricoides/embriologia , Ascaris lumbricoides/metabolismo , Sequência de Bases , Modelos Genéticos , Dados de Sequência Molecular , Mutação , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , Spliceossomos/metabolismo , Relação Estrutura-Atividade , TionucleotídeosRESUMO
We have used block-substitution mutagenesis and in vitro transcription assays to identify cis-acting DNA sequence elements important for initiation and 3'-end formation of a U1 small nuclear RNA (snRNA) in the parasitic nematode Ascaris lumbricoides. Efficient initiation of synthesis by RNA polymerase II requires a compact element centered approximately 50 base pairs upstream of the transcriptional start site. Surprisingly, 3'-end formation of U1 snRNAs synthesized in vitro is solely dependent upon elements within the U1 coding sequence. In all other systems studied thus far, 3'-end formation of U snRNAs requires signals present in the 3'-flanking region. We also show that sequence elements that direct 3'-end formation of the A. lumbricoides trans-spliced leader RNA function when RNA synthesis is initiated from the U1 promoter. These results indicate that 3'-end formation of U snRNAs in nematodes is mechanistically distinct from the analogous process in higher eukaryotes.
Assuntos
Ascaris lumbricoides/genética , RNA Nuclear Pequeno/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Animais , Sequência de Bases , DNA , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Maturation of nuclear pre-mRNAs in nematodes requires both cis- and trans-splicing. Both processing pathways involve analogous two-step phosphotransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional reconstitution assay to assess the U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. The frequency of this event was greatly enhanced when a highly conserved sequence in U6 snRNA was altered by mutation. In cis- and trans-splicing reactions reconstituted with this mutant U6 snRNA the liberated exon of U6 proceeded through the second step of splicing using the appropriate splice acceptor sites. These results demonstrate covalent interactions between a U snRNA required for splicing and a splicing substrate, and they provide evidence for an unexpected degree of catalytic flexibility within the spliceosome.
Assuntos
Ascaris/metabolismo , Éxons , Splicing de RNA , RNA Nuclear Pequeno/biossíntese , Animais , Ascaris/embriologia , Composição de Bases , Sequência de Bases , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/isolamento & purificação , Spliceossomos/metabolismoRESUMO
Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing.
Assuntos
Ascaris/genética , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Éxons , Modelos Estruturais , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genéticaRESUMO
In humans, the H1 RNA, the RNA subunit of RNase P, is synthesized by RNA polymerase III. We have used block replacement mutagenesis to identify the sequences necessary for in vitro transcription of H1 RNA. We find that multiple cis-acting elements located in the H1 RNA 5'-flanking region are necessary for H1 RNA synthesis; no internal sequences are essential. Required cis-acting elements include sequences resembling proximal sequence element, distal sequence element, and TATA motifs. In this respect, the H1 RNA promoter is similar in structure to the promoters of the genes encoding the U6 snRNA, the 7 SK RNA and the MRP RNA. However, our mutational analysis indicates that the H1 promoter is unexpectedly complex, with several additional cis-acting elements spanning nearly 70 base pairs of the H1 RNA gene 5'-flanking sequence.
Assuntos
Endorribonucleases/genética , Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA Catalítico/genética , RNA/genética , Transcrição Gênica , Sequência de Bases , DNA , Endorribonucleases/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA Catalítico/metabolismo , Ribonuclease P , Moldes GenéticosRESUMO
The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.
Assuntos
Composição de Bases , Nematoides/genética , Sinais Direcionadores de Proteínas/metabolismo , Splicing de RNA , Animais , Sequência de Bases , Éxons , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismoRESUMO
In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5'-terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis- and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis- and trans-splicing. These experiments indicated that both cis- and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (snRNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, trans-splicing is unaffected when approximately 90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis- and trans-splicing.
Assuntos
Splicing de RNA , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Ascaris , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Ribonucleoproteínas Nucleares PequenasRESUMO
The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure.
Assuntos
Ascaris/genética , Splicing de RNA , Ribonucleoproteínas/fisiologia , Animais , Autoantígenos/fisiologia , Sequência de Bases , Sítios de Ligação , Análise Mutacional de DNA , Técnicas In Vitro , Dados de Sequência Molecular , Precursores de Ácido Nucleico/metabolismo , Oligonucleotídeos , Capuzes de RNA , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas , Proteínas Centrais de snRNPRESUMO
Immunization of mice with irradiated Brugia larvae or parasite extracts has been shown to induce partial resistance to microfilaremia and enhance clearance of infective larvae. We recently reported the cloning of a 548 amino acid 62-kDa Brugia malayi Ag identified on the basis of reactivity with antisera to a subset of protective microfilarial Ag. Our study describes the protective efficacy against microfilaremia in mice, immunogenicity, and parasite stage-specificity of this candidate vaccine molecule. Immunization of Swiss or BALB/c mice with 1 to 3 micrograms of a 92-kDa trpE fusion protein encoding amino acids 1-479 reduced the intensity of microfilaremia by 40 to 60% compared to control animals given buffer or bacterial trpE (p less than 0.01 to 0.001). Mice immunized with the 92-kDa fusion protein developed delayed-type hypersensitivity reactivity to B. malayi as assessed by enhanced footpad swelling 24 and 48 h after intradermal injection of adult worm extract and in vitro lymph node mononuclear cell proliferation (3H-thymidine uptake) in response to the fusion protein (mean +/- SD stimulation index 4.7 +/- 0.8 vs 2.0 +/- 1.4 for trpE, p less than 0.05). Proliferative responses of lymph node cells coincubated with three other fusion proteins corresponding to the filarial protein truncated from its carboxyl-terminus suggest that dominant T cell epitopes of the 62-kDa Ag are encompassed by amino acids 437-479. Rabbit antibody to the 92-kDa trpE fusion protein immunoprecipitated a 62-kDa polypeptide from [35S] methionine biosynthetically labeled B. malayi microfilariae, adult female, and adult male worms. These data indicate that a recombinant Ag expressed in several developmental stages of B. malayi is capable of inducing partial resistance against microfilariae and Ag-specific T cell responses in mice.
Assuntos
Antígenos de Helmintos/imunologia , Brugia/imunologia , Filariose Linfática/prevenção & controle , Filariose/prevenção & controle , Animais , Formação de Anticorpos , Antígenos de Helmintos/análise , Antígenos de Helmintos/genética , Clonagem Molecular , DNA/genética , Filariose Linfática/imunologia , Filariose Linfática/parasitologia , Ativação Linfocitária , Camundongos , Peso Molecular , Proteínas Recombinantes de Fusão/imunologia , Testes Cutâneos , Linfócitos T/imunologia , Vacinas SintéticasRESUMO
In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.
Assuntos
Ascaris/genética , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Sequência de Bases , Endorribonucleases , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Sondas de Oligonucleotídeos , Ribonuclease H , Ribonuclease T1RESUMO
We have used block substitution mutagenesis and in vitro transcription to define sequence elements important for efficient initiation and 3' end-formation of the trans-spliced leader RNA (SL RNA) of the parasitic nematode Ascaris lumbricoides. These experiments indicate that the SL RNA has an unusual promoter structure containing elements which include the 22 nt trans-spliced leader exon itself. Efficient transcription is correlated with the binding of a factor to the 22 nt (SL) sequence; mutations within the SL which abolish transcription lead to a loss in binding of this factor. In addition to internal sequences, synthesis of SL RNA in vitro requires an element centered 50 bases upstream of the cap site. Mutations within this element dramatically affect the level of SL RNA synthesis but do not affect accuracy of initiation. Finally, all of the information required for accurate 3' end-formation of SL RNA lies within the transcribed region. Thus, the arrangement of sequences necessary for the synthesis of SL RNAs does not resemble that of sequences important for the synthesis of vertebrate U snRNAs despite the similarities between SL RNAs and U snRNAs.
Assuntos
Ascaris/genética , Splicing de RNA , RNA Mensageiro/biossíntese , Transcrição Gênica , Animais , Sequência de Bases , Desoxirribonuclease I , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Mapeamento por RestriçãoRESUMO
Maturation of a fraction of mRNAs in nematodes involves the acquisition of a common 5' terminal spliced leader sequence derived from a nonpolyadenylylated spliced leader RNA by trans splicing. We have developed a cell-free system prepared from Ascaris lumbricoides embryos that accurately and efficiently synthesized the spliced leader RNA of A. lumbricoides. Transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethylguanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA.
Assuntos
Ascaris/genética , Capuzes de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Transcrição Gênica , Animais , Ascaris/embriologia , Sequência de Bases , Sistema Livre de Células , Embrião não Mamífero/metabolismo , Metilação , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Capuzes de RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Nuclear Pequeno/genéticaRESUMO
We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.