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PubMed1997

Hepatocarcinogenesis in woodchuck hepatitis virus/c-myc mice: sustained cell proliferation and biphasic activation of insulin-like growth factor II.

Transgenic mice carrying the c-myc oncogene under control of woodchuck hepatitis virus (WHV) DNA sequences invariably develop hepatocellular carcinoma (HCC), despite a temporally limited expression of the transgene in the neonatal liver. To better characterize the different steps of the tumorigenic process, we analyzed the liver expression of the c-myc transgene and several growth-related genes by in situ hybridization and Northern blotting. In parallel studies, proliferated changes were investigated by detection of bromodeoxy-uridine-positive S-phase nuclei and apoptosis was evaluated by in situ nick end-labeling of DNA. During the neonatal period, high levels of c-myc messenger RNAs (mRNAs) were detected in all hepatocytes, and the expression of insulin-like growth factor II (IGF II) was frequently enhanced, correlating with increased cell proliferation. Despite elevated expression of the p53 gene, no change in liver cell apoptosis was observed. After weaning, c-myc transgene expression decreased to undetectable levels in all hepatocytes, whereas proliferation decreased but remained notably higher than in age-matched controls. The expression of c-fos, c-jun, and c-H-ras was highly variable during the preneoplastic period and in the tumors, with no consistent increase compared with controls. Resurgence of c-myc transgene expression was evidenced in all cells from hyperplastic lesions and carcinomas, accompanied with frequent focal reactivation of IGF II. Thus the strong proliferative stimulus induced by the combined effects of c-myc and IGF II in the neonatal liver might initiate a process characterized by persistent, dysregulated hepatocyte proliferation, in turn greatly increasing the risk of hepatocellular transformation.

Animals↗

PubMed1988

Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures.

This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

Amino Acids↗

PubMed2006

Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi.

Argonaute (AGO) proteins interact with small RNAs to mediate gene silencing. C. elegans contains 27 AGO genes, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Here we describe 31 deletion alleles representing all of the previously uncharacterized AGO genes. Analysis of single- and multiple-AGO mutant strains reveals functions in several pathways, including (1) chromosome segregation, (2) fertility, and (3) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other AGO proteins interact with amplified siRNAs to mediate downstream silencing. Overexpression of downstream AGOs enhances silencing, suggesting that these proteins are limiting for RNAi. Interestingly, these AGO proteins lack key residues required for mRNA cleavage. Our findings support a two-step model for RNAi, in which functionally and structurally distinct AGOs act sequentially to direct gene silencing.

Amino Acid Sequence↗

PubMed2000

Effects of cetyltrimethylammonium bromide on reactions catalyzed by maxizymes, a novel class of metalloenzymes.

We demonstrated previously that some shortened forms of hammerhead ribozymes had high cleavage activity that was similar to that of the wild-type parental hammerhead ribozyme. Moreover, the active species appeared to form dimeric structures with a common stem II (in order to distinguish monomeric forms of conventional minizymes that have low activity from our novel dimers with high-level activity, the latter very active short ribozymes were designated 'maxizymes'). The dimers can be homodimeric (with two identical binding sequences) or heterodimeric (with two different binding sequences). In the case of heterodimers, they are in equilibrium with inactive homodimers. In this study, we investigated the effects of cationic detergent, cetyltrimethylammonium bromide (CTAB), on reactions catalyzed by a variety of maxizymes. The slope of close to unity in profiles of pH versus rate demonstrated that the deprotonation was important in catalysis and that the rate-limiting chemical step was followed in these reactions. Addition of appropriate amounts of CTAB enhanced the activity of a variety of maxizymes. The activity of our least stable, least active maxizyme was enhanced 100-fold by CTAB. Thus, CTAB effectively enhanced the conversion of kinetically trapped inactive conformations to active forms. Moreover, we suggest that the activity and specificity of catalytic RNAs in vivo might be better estimated if their reactions are monitored in vitro in the presence of appropriate amounts of CTAB.

Base Pairing↗

PubMed1990

Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours.

The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.

Amino Acid Sequence↗

PubMed2005

RNA interference is an antiviral defence mechanism in Caenorhabditis elegans.

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing mechanism that is well defined genetically in Caenorhabditis elegans. RNAi has been postulated to function as an adaptive antiviral immune mechanism in the worm, but there is no experimental evidence for this. Part of the limitation is that there are no known natural viral pathogens of C. elegans. Here we describe an infection model in C. elegans using the mammalian pathogen vesicular stomatitis virus (VSV) to study the role of RNAi in antiviral immunity. VSV infection is potentiated in cells derived from RNAi-defective worm mutants (rde-1; rde-4), leading to the production of infectious progeny virus, and is inhibited in mutants with an enhanced RNAi response (rrf-3; eri-1). Because the RNAi response occurs in the absence of exogenously added VSV small interfering RNAs, these results show that RNAi is activated during VSV infection and that RNAi is a genuine antiviral immune defence mechanism in the worm.

Animals↗

PubMed2001

Alternative splicing of the imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and DNA binding activities.

ZAC encodes a zinc finger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZACDelta2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal zinc fingers. Messenger RNAs encoding ZAC or ZACDelta2 were equally abundant and both proteins were nuclear. ZACDelta2 displayed an improved transactivation activity and an enhanced affinity for a ZAC binding site, suggesting that the two N-terminal zinc fingers negatively regulated ZAC binding to its target DNA sequences. Both proteins were equally efficient in preventing colony formation, indicating similar overall antiproliferative activities. However, these activities resulted from a differential regulation of apoptosis vs cell cycle progression since ZACDelta2 was more efficient at induction of cell cycle arrest than ZAC, whereas it was the reverse for apoptosis induction. Hence, these data further underline that ZAC gene is critically controlled, both at the transcriptional level through imprinting and at the functional level through alternative splicing.

Alternative Splicing↗

PubMed1999

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

Adaptor Proteins, Signal Transducing↗

PubMed1992

Selective cleavage of closely-related mRNAs by synthetic ribozymes.

In Phaseolus vulgaris L. (French bean) glutamine synthetase (GS) is encoded by four closely-related genes termed gln-alpha, gln-beta, gln-gamma and gln-delta. We have constructed and characterised in vitro a number of hammerhead ribozymes designed to cleave individual RNAs encoded by these genes. The three ribozymes, termed J1, J2 and J3, were targeted to cleave RNA at the start of the gamma and beta, and the middle of the gamma, GS open reading frames respectively. All three ribozymes successfully discriminated between the four (alpha, beta, gamma and delta) highly homologous sequences, even though the targeted sites of cleavage shared up to 18 out of 22 identical bases with other gene family members. The ribozyme-mediated cleavage reactions were Mg2+ dependent and enhanced at higher temperatures, although the J1 ribozyme retained considerable activity at physiological temperatures. Both J1 and J2 demonstrated a time-dependent cleavage of their targeted GS RNAs, although these two ribozymes differed markedly in their ability to cleave multiple substrate molecules. The rate of cleavage by J1 was found to be reduced in the presence of related GS RNAs and by total leaf poly(A) RNAs. The implications of these results for ribozyme activity in vivo are discussed.

Amino Acid Sequence↗

PubMed2004

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin.

Discrete sequence elements known as exonic splicing enhancers (ESEs) have been shown to influence both the efficiency of splicing and the profile of mature mRNAs in multicellular eukaryotes. While the existence of ESEs has not been demonstrated previously in unicellular eukaryotes, the factors known to recognize these elements and mediate their communication with the core splicing machinery are conserved and essential in the fission yeast Schizosaccharomyces pombe. Here, we provide evidence that ESE function is conserved through evolution by demonstrating that three exonic splicing enhancers derived from vertebrates (chicken ASLV, mouse IgM, and human cTNT) promote splicing of two distinct S. pombe pre-messenger RNAs (pre-mRNAs). Second, as in extracts from mammalian cells, ESE function in S. pombe is compromised by mutations and increased distance from the 3'-splice site. Third, three-hybrid analyses indicate that the essential SR (serine/arginine-rich) protein Srp2p, but not the dispensable Srp1p, binds specifically to both native and heterologous purine-rich elements; thus, Srp2p is the likely mediator of ESE function in fission yeast. Finally, we have identified five natural purine-rich elements from S. pombe that promote splicing of our reporter pre-mRNAs. Taken together, these results provide strong evidence that the genesis of ESE-mediated splicing occurred early in eukaryotic evolution.

Animals↗

PubMed2025

Proteomic profiling identifies miR-423-5p as a modulator of oncogenic metabolism in HCC.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a significant clinical challenge due to limited diagnostic and therapeutic options. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), play key roles in cancer biology. Our previous findings showed that miR-423-5p enhances anti-cancer effects on HCC patients treated with sorafenib by promoting autophagy. Here, we investigated the molecular mechanisms underlying miR-423-5p function through a comprehensive proteomic approach. METHODS: We generated an HCC cell line stably overexpressing miR-423-5p via lentiviral transduction. Total proteins were extracted from SNU-387 cells, enzymatically digested into peptides, and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/M). Raw spectral data were processed and quantified using MaxQuant. Differentially expressed proteins (DEPs) were defined based on fold-change (|log2FC| ≥ 1) and false discovery rate (FDR < 0.05). The full proteomic dataset is available via the ProteomeXchange repository (identifier: PXD064869). Functional enrichment analysis of DEPs were performed using DAVID and Reactome. To assess clinical relevance, predicted and validated miR-423-5p targets were integrated with The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) dataset using GEPIA platform. Survival analyses were performed using the Kaplan-Meier method. RESULTS: Proteomic profiling identified 698 DEPs in miR-423-5p-overexpressing cells compared to controls with significant enrichment in metabolic pathways, related to purine/pyrimidine metabolism and gluconeogenesis. Integration with bioinformatic predictions and miRTarBase validation identified 43 DEPs as potential direct targets of miR-423-5p. Among these, seven proteins (ACACA, ANKRD52, DVL3, MCM5, MCM7, RRM2, SPNS1, and SRM) were significantly associated with patient prognosis in the TCGA-LIHC cohort. These targets were downregulated in miR-423-5p-overexpressing cells but upregulated in advanced-stage HCC tissues, suggesting a potential role for miR-423-5p in the regulation of HCC pathogenesis. Stage-specific expression analysis showed increased levels from stage I to III, followed by a decline at stage IV. Notably, we experimentally confirmed miR-423-5p-mediated suppression of MCM7, DVL3, IMPDH1, and SRM (SPEE), supporting their functional involvement in HCC progression. CONCLUSION: Overall, our findings support a tumour-suppressive role for miR-423-5p in HCC, mediated by modulation of metabolic pathways and suppression of oncogenic proteins. These results suggest that miR-423-5p and its downstream effectors may serve as promising biomarkers and potential therapeutic targets in HCC. HIGHLIGHTS: miR-423-5p acts as a tumor suppressor in HCC by targeting key nodes of pro-tumorigenic signalling. miR-423-5p significantly altered metabolic pathways, including purine/pyrimidine metabolism and gluconeogenesis. Seven miR-423-5p targets correlate with poor prognosis in TCGA-LIHC patients and are downregulated in miR-423-5p overexpressing HCC cells. miR-423-5p over-expression induces a significant downregulation of MCM7, DVL3, IMPDH1, SPEE in HCC cell models. miR-423-5p limits tumor metabolic plasticity, suggesting therapeutic potential.

MicroRNAs↗

PubMed2002

Multiple, conserved iron-responsive elements in the 3'-untranslated region of transferrin receptor mRNA enhance binding of iron regulatory protein 2.

Synthesis of proteins for iron homeostasis is regulated by specific, combinatorial mRNA/protein interactions between RNA stem-loop structures (iron-responsive elements, IREs) and iron-regulatory proteins (IRP1 and IRP2), controlling either mRNA translation or stability. The transferrin receptor 3'-untranslated region (TfR-3'-UTR) mRNA is unique in having five IREs, linked by AU-rich elements. A C-bulge in the stem of each TfR-IRE folds into an IRE that has low IRP2 binding, whereas a loop/bulge in the stem of the ferritin-IRE allows equivalent IRP1 and IRP2 binding. Effects of multiple IRE interactions with IRP1 and IRP2 were compared between the native TfR-3'-UTR sequence (5xIRE) and RNA with only 3 or 2 IREs. We show 1) equivalent IRP1 and IRP2 binding to multiple TfR-IRE RNAs; 2) increased IRP-dependent nuclease resistance of 5xIRE compared with lower IRE copy-number RNAs; 3) distorted TfR-IRE helix structure within the context of 5xIRE, detected by Cu-(phen)(2) binding/cleavage, that coincides with ferritin-IRE conformation and enhanced IRP2 binding; and 4) variable IRP1 and IRP2 expression in human cells and during development (IRP2-mRNA predominated). Changes in TfR-IRE structure conferred by the full length TfR-3'-UTR mRNA explain in part evolutionary conservation of multiple IRE-RNA, which allows TfR mRNA stabilization and receptor synthesis when IRP activity varies, and ensures iron uptake for cell growth.

3' Untranslated Regions↗

PubMed1989

The Epstein-Barr virus (EBV) DR enhancer contains two functionally different domains: domain A is constitutive and cell specific, domain B is transactivated by the EBV early protein R.

The Epstein-Barr virus (EBV) DR promoter is located upstream of the PstI repeats, and besides the TATA box, it contains two cis-acting regulatory elements. One of them has enhancer properties. To define more precisely the functional region(s) in the DR enhancer, we generated 5' and 3' deletion mutants. These deletion mutants, which were transfected into various recipient cells of different origins, allowed us to identify two functionally distinct domains, A and B. Domain A was constitutively active in all cell lines tested, except in lymphoid B cells. Domain B was active in lymphoid B cells, and its activity required both EB1 (the BZLF1-encoded EBV trans-acting factor) and the presence of the EBV genome. This suggested that an EBV-encoded, EB1-inducible factor was activating the enhancer B domain. In effect, the B domain was trans-activated by R, an EBV early product encoded by the open reading frame BRLF1, and the activation by R occurred in epithelial, fibroblastic, and lymphoid cells. The R-responsive element has been reduced to 28 base pairs containing the double palindromic sequence TTGTCCCGTGGACAATGTCC. Both domains A and B act by increasing the initiation of specific RNAs.

Animals↗

PubMed2004

Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice.

Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Animals↗

PubMed1995

Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity.

A functional analysis of exon replacement mutations was performed in parallel with RNA-protein binding assays to gain insight into the role of the exon in alternative and simple splicing events. These results show that constitutive exons from unrelated genes contain strong signals that promote splicing in multiple sequence contexts by enhancing 3' splice site activity. A clue to the nature of the relationship between the exon and adjacent 3' splice site is indicated by the binding properties of exon variant RNAs when tested with different biochemical preparations of the essential splicing protein, U2AF. In the context of a complete nuclear extract, U2AF binding to the 3' splice site is stimulated by the presence of an adjacent constitutive exon. In contrast, highly purified HeLa U2AF binds equivalently to the exon variants under conditions in which differential polypyrimidine tract binding is evident. These results provide support for an assisted binding model in which positive-acting signals within exons, exon enhancers, direct the binding of accessory factors, which in turn increase the intrinsic affinity of U2AF for the adjacent 3' splice site. Further support for an assisted binding model is indicated by biochemical complementation of U2AF binding and by the localization of a novel exon enhancer, which, when introduced into a weak exon, stimulates splicing activity in parallel with U2AF binding. Immunoprecipitation analysis identifies the splicing factor, SC35, as a constituent of the exon enhancer binding complex. These results are discussed in the context of current models for functional exon-bridging interactions.

Base Sequence↗

PubMed2006

DDX3, a DEAD box RNA helicase, is deregulated in hepatitis virus-associated hepatocellular carcinoma and is involved in cell growth control.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide and is highly correlated with hepatitis virus infection. Our previous report shows that a DEAD box RNA helicase, DDX3, is targeted and regulated by hepatitis C virus (HCV) core protein, which implicates the involvement of DDX3 in HCV-related HCC development. In this study, the potential role of DDX3 in hepatocarcinogenesis is investigated by examining its expression in surgically excised human HCC specimens. Here we report the differential deregulation of DDX3 expression in hepatitis virus-associated HCC. A significant downregulation of DDX3 expression is found in HCCs from hepatitis B virus (HBV)-positive patients, but not from HCV-positive ones, compared to the corresponding nontumor tissues. The expression of DDX3 is differentially regulated by the gender and, moreover, there is a tendency that the downregulation of DDX3 expression in HCCs is more frequent in males than in females. Genetic knockdown of DDX3 with small interfering RNAs (siRNA) in a nontransformed mouse fibroblast cell line, NIH-3T3, results in a premature entry to S phase and an enhancement of cell growth. This enhanced cell cycle progression is linked to the upregulation of cyclin D1 and the downregulation of p21(WAF1) in the DDX3 knockdown cells. In addition, constitutive reduction of DDX3 expression increases the resistance of NIH-3T3 cells to serum depletion-induced apoptosis and enhances the ras-induced anchorage-independent growth, indicating the involvement of DDX3 in cell growth control. These findings together with the previous study suggest that the deregulation of DDX3, a DEAD box RNA helicase with cell growth-regulatory functions, is involved in HBV- and HCV-associated pathogenesis.

Animals↗

PubMed1995

Viomycin does not stimulate the dissociation of peptidyl-tRNA.

Peptidyl-transfer RNA normally dissociates at a low rate from the ribosomes of Escherichia coli during protein synthesis but accumulates under nonpermissive conditions in cells with a temperature-sensitive allele (pthts) of the gene encoding peptidyl-transfer RNA hydrolase. The antibiotic-hypersensitive strain E. coli DB-11 with the pthts mutation was exposed to viomycin, then placed at nonpermissive temperatures. Under these conditions in the absence of drugs, peptidyl-tRNA accumulates, protein synthesis is inhibited and pthts cells die. When viomycin was present at sufficient concentration to arrest protein synthesis, cell death was not accelerated, error-inducing effects of streptomycin were not counteracted and, at high doses, cytoplasmic accumulation of peptidyl-transfer RNA was slowed down. Blocking the translocation of peptidyl-transfer RNA with viomycin did not stimulate its dissociation from ribosomes. Erythromycin-enhanced cell death was not affected by viomycin at doses sufficient to block amino acid incorporation, suggesting that short peptidyl-transfer RNAs could still be synthesized and dissociated from ribosomes.

Drug Combinations↗

PubMed1994

Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.

To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.

Amino Acid Sequence↗