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Results for “RNA 3' End Processing”

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At least 19 recordsLinked to original sources

PubMed2025

Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3' end processing in polycistronic mRNA.

"Pre-full-length" transcripts are produced at the end of the polycistronic galactose (gal) operon, 5' galE-galT-galK-galM 3', via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3' end of the full-length galETKM mRNA is acquired by exonucleolytic processing of the 3'-OH ends of the pre-full-length transcripts. However, the gal operon produces an mRNA termed galE whose 3' end forms approximately 120 nucleotides downstream of the galE stop codon, within the subsequent gene, galT, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3' end of galE mRNA. We discovered that the 3' ends of pre-galE mRNA are produced in the middle of galT as a result of the combination of two separate molecular processes-one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3' ends of pre-galE mRNA undergo exonucleolytic processing to the 3' end of galE mRNA observed in vivo. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5-10 nucleotides upstream of the 3' ends of galE mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E-contrary to its general role in mRNA degradation-produces RNA 3' ends that regulate polarity in gene expression.IMPORTANCEThis study reports the findings of two molecular mechanisms that generate the 3' ends of pre-galE mRNA in the gal operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3' ends are subsequently processed to produce stable galE mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3' end of galE mRNA within galT in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.

RNA, Messenger↗

PubMed1992

Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing.

We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992).

Animals↗

PubMed2025

Decoding ALS from the tail end of RNA.

In this issue of Cell Genomics, McKeever et al.1 generate a single-nucleus transcriptomic atlas of ALS/FTLD brain and reveal widespread alternative polyadenylation changes. Their findings highlight 3' end RNA processing as a central integrator of stress responses, cell-type specificity, and disease susceptibility, offering new mechanistic insight and potential therapeutic directions.

Cell Nucleus↗

PubMed1992

A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.

The Neurospora crassa cyt-4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' end processing, and RNA turnover. Here, we show that the cyt-4+ gene encodes a 120-kDa protein with significant similarity to the SSD1/SRK1 protein of Saccharomyces cerevisiae and the DIS3 protein of Schizosaccharomyces pombe, which have been implicated in protein phosphatase functions that regulate cell cycle and mitotic chromosome segregation. The CYT-4 protein is present in mitochondria and is truncated or deficient in two cyt-4 mutants. Assuming that the CYT-4 protein functions in a manner similar to the SSD1/SRK1 and DIS3 proteins, we infer that the mitochondrial RNA splicing and processing reactions defective in the cyt-4 mutants are regulated by protein phosphorylation and that the defects in the cyt-4 mutants result from failure to normally regulate this process. Our results provide evidence that RNA splicing and processing reactions may be regulated by protein phosphorylation.

Amino Acid Sequence↗

PubMed1992

Two distant upstream regions containing cis-acting signals regulating splicing facilitate 3'-end processing of avian sarcoma virus RNA.

Retroviruses, pararetroviruses, and related retrotransposons generate terminally redundant RNAs by transcription of a template flanked by long terminal repeats in which initiation occurs within the 5' long terminal repeat sequences and 3'-end processing occurs within the 3' long terminal repeat sequences. Processing of avian sarcoma virus RNA is relatively inefficient; approximately 15% of the viral RNA transcripts are read-through products; i.e., they are not processed at the viral poly(A) addition site but at sites in the cellular sequence further downstream. In this report, we show that the efficiency of processing at the viral site is further reduced by deletion of two distant upstream sequences: (i) a 606-nucleotide sequence in the gag gene containing a cis-acting negative regulator of splicing and (ii) a 136-nucleotide sequence spanning the env 3' splice site. The deletion of either or both upstream regions increases the levels of read-through products of both unspliced and spliced viral RNA. In contrast, deletion of the src 3' splice site does not affect the efficiency of processing at the viral poly(A) addition site. The effects on 3'-end processing are not correlated either with distance from the promoter to the poly(A) addition site or with the overall level of viral RNA splicing. Substitution of the avian sarcoma virus poly(A) signal with the simian virus 40 early or late poly(A) signal relieves the requirement for the distant upstream sequences. We propose that cellular factors, which may correspond to splicing factors, bound to the upstream viral sequences may interact with factors bound at the avian sarcoma virus poly(A) signal to stabilize the polyadenylation-cleavage complex and allow for more efficient 3'-end processing.

Animals↗

PubMed2005

Inactivation of the pre-mRNA cleavage and polyadenylation factor Pfs2 in fission yeast causes lethal cell cycle defects.

Faithful chromosome segregation is fundamentally important for the maintenance of genome integrity and ploidy. By isolating conditional mutants defective in chromosome segregation in the fission yeast Schizosaccharomyces pombe, we identified a role for the essential gene pfs2 in chromosome dynamics. In the absence of functional Pfs2, chromosomal attachment to the mitotic spindle was defective, with consequent chromosome missegregation. Under these circumstances, multiple intracellular foci of spindle checkpoint proteins Bub1 and Mad2 were seen, and deletion of bub1 exacerbated the mitotic defects and the loss of cell viability that resulted from the loss of pfs2 function. Progression from G1 into S phase following release from nitrogen starvation also required pfs2+ function. The product of the orthologous Saccharomyces cerevisiae gene PFS2 is a component of a multiprotein complex required for 3'-end cleavage and polyadenylation of pre-mRNAs and, in keeping with the conservation of this essential function, an S. pombe pfs2 mutant was defective in mRNA 3'-end processing. Mutations in pfs2 were suppressed by overexpression of the putative mRNA 3'-end cleavage factor Cft1. These data suggest unexpected links between mRNA 3'-end processing and chromosome replication and segregation.

Carrier Proteins↗

PubMed1992

Analysis of cauliflower mosaic virus RNAs in Brassica species showing a range of susceptibility to infection.

Cauliflower mosaic virus (CaMV) is a plant pararetrovirus i.e., a DNA virus that replicates through reverse transcription of its terminally redundant genomic RNA (the 35 S RNA). In this study, the absolute levels and relative ratios of CaMV-encoded RNA species were analyzed in Brassica host plants with different susceptibilities to infection. As reported previously, only very low levels of CaMV RNAs were detected in plants of low susceptibility such as cauliflower. Early in infection, a large proportion of these RNAs were the "short-stop" RNA: a 180-nucleotide RNA generated by mRNA 3' end processing at the first encounter of the polyadenylation [poly(A)] signal rather than at the second encounter by which the 35 S RNA is generated. In contrast, in highly susceptible plants such as turnip, high levels of CaMV RNAs were detected, and the short-stop RNA represented only a small fraction of the RNA. In leaf protoplasts, bypass of the poly(A) signal was similar in all Brassica species. Finally, the ratio of the 19 S RNA, a subgenomic RNA encoding a post-transcriptional trans-activator, to the 35 S RNA was lower in cauliflower than in turnip. These results are discussed in light of the CaMV life cycle.

Brassica↗

PubMed1992

A method for generating transcripts with defined 5' and 3' termini by autolytic processing.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

Autolysis↗

PubMed1992

An exonuclease requiring an intact helical stem for specificity produces the 3' end of Acanthamoeba castellanii 5 S RNA.

A nuclear extract from Acanthamoeba castellanii which contains all of the components necessary for specific transcription of a 5 S RNA gene was separated into fractions required for specific transcription initiation and an additional fraction needed in the reconstituted system to produce the 3' end characteristic of mature 5 S RNA. The latter fraction contained a novel processing activity characterized by an exonuclease specific for highly structured RNAs, including 5 S RNA. An intact helical stem formed between the 5' and 3' ends of the 5 S RNA precursor determines the 3' nucleotide. In addition, the presence of ATP is required for specific processing. However, the possibility has not been ruled out that ATP inhibits a nonspecific ribonuclease in the extract since processing proceeds into the helical stem in its absence.

Acanthamoeba↗

PubMed1992

Characterization of two types of ribosomal gene transcription in Xenopus laevis oocytes.

When the germinal vesicle of Xenopus laevis oocytes is translocated into the vegetal hemisphere by centrifugation, the normally silent ribosomal spacer promoters are strongly induced. This induction correlates with the permeability of the nuclear envelope to dextran of molecular weight 70 kDa, thus raising the possibility that the transcriptional changes are due to mixing of nuclear and cytoplasmic components. This basic observation prompted a thorough investigation of ribosomal gene transcription in centrifuged oocytes which had the germinal vesicle either in the animal half (A-oocytes) or in the vegetal half (V-oocytes). Two types of ribosomal gene transcription were characterized: (1) in A-oocytes, spacer promoters remain silent, transcription initiation is dependent on the upstream terminator T3, and transcription is highly processive and recognizes sites of RNA 3' end formation (like T2 and T3); (2) in V-oocytes, spacer promoters are induced, transcription initiation is independent of T3, but most transcripts terminate prematurely after less than 150 nt. Furthermore, the transcription machinery in V-oocytes does not respond to T2 or T3 signals. The implications of the present observations for our understanding of the regulation of the spacer promoters and of the function of the upstream terminator T3 are discussed.

Animals↗

PubMed1992

The effects of stem I and loop A on the processing of 5 S rRNA from Drosophila melanogaster.

The 135-nucleotide Drosophila melanogaster 5 S RNA precursor is processed by removal of 15 nucleotides from its 3' end before incorporation into the large ribosomal subunit. Mature 5 S RNA consists of five helical stem-loops; stem IV and part of V are dispensable, whereas stem III and the 1/118 G-C base pair closest to the processing site at nucleotide 120 are required for processing (Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 7509-7516; Preiser, P., and Levinger, L. (1991) J. Biol. Chem. 266, 23602-23605). We have investigated the effects of stem I and loop A transversions, transitions, selected additions and deletions on 5 S RNA processing. Stem I single substitutions generally prevent processing, whereas compensatory double substitutions restore a range of processing rates. Proximal to the processing site, stem I double substitutions inhibit processing. In the distal portion of stem I and loop A, the processing effect of paired sequence changes varies widely in an irregular pattern. The 7/112 GU pair and nucleotide 13A least tolerate sequence changes; several mutations clustered close to the stem I-loop A boundary stimulate processing. We interpret these results in terms of the RNA helix path and possible RNA-protein contacts.

Animals↗

PubMed2023

Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3.

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.

Cleavage And Polyadenylation Specificity Factor↗

PubMed1992

Nuclear processing of the 3'-terminal nucleotides of pre-U1 RNA in Xenopus laevis oocytes.

U1 small nuclear RNA is synthesized as a precursor with several extra nucleotides at its 3' end. We show that in Xenopus laevis oocytes, removal of the terminal two nucleotides occurs after the RNA has transited through the cytoplasm and returned to the nucleus. The activity is controlled by an inhibitor of processing, which we call TPI, for 3'-terminal processing inhibitor. This inhibitor is sensitive to both micrococcal nuclease and trypsin treatment, indicating that it is a nucleoprotein. TPI inhibits the 3' processing of pre-U1 RNAs that have 5' ends containing m7G caps but not mature m2,2,7G caps; this finding suggests that TPI interacts directly or indirectly with the 5' end of pre-U1 RNA. The inhibition of processing by TPI, almost complete at 19 degrees C, is reversibly inactivated at slightly higher temperatures. TPI activity is solely in the soluble fraction of oocyte nuclear extracts, in contrast to the 3'-terminal processing activity, which is present in both the particulate and soluble fractions. We propose that the differential processing of the 3'-terminal nucleotides of pre-U1 RNA after its return from the cytoplasm, but not before its exit from the nucleus, may be due to the association of TPI with the m7G cap on the newly synthesized pre-U1 RNA.

Animals↗

PubMed1992

Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae.

A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements. Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA. Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition. Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed. We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme. By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing. This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose. As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro. Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.

Base Sequence↗

PubMed1992

Cyclic 2',3'-phosphates and nontemplated nucleotides at the 3' end of spliceosomal U6 small nuclear RNA's.

Spliceosomal U6 small nuclear RNA (U6 RNA) in species as diverse as man, frog, fruitfly, and soybean have at their 3' ends a cyclic 2',3'-phosphate (greater than p) apparently derived from uridylic acid residues that were added post-transcriptionally. The 3' ends of U6 RNA's from various sources may be processed in different ways, or to different extents, depending on the organism or stage of development. The presence of a greater than p terminus on U6 RNA may influence the activity of U6 RNA either directly during splicing or indirectly by ensuring that the RNA has a defined length or proper conformation (or both).

Animals↗

PubMed1976

Structural organization and processing of the genetic transcript in the cellular slime mold Dictyostelium discoideum.

The organization of the genome and the synthesis and processing of heterogeneous nuclear RNA (HNRNA) in the cellular slime mold Dictysotelium discoideum have been analyzed. Approximately 60-70% of the genome of Dictyostelium consists of interspersed reiterated and single-copy sequences. The interspersed reiterated sequences have an average length of 250-400 nucleotides. Approximately 50% of the reiterated DNA sequences consist of long noninterspersed sequences. The results of analyses of ynRNA synthesis and processing have been incorporated into a model. According to the model the primary genetic transcript of Dictyostelium is synthesized as a molecule that is 25% larger than mRNA. The bulk of the hnRNA is synthesized from a unit consisting of a short reiterated DNA sequence transcript at the 5' end of the molecule and a single-copy sequence of approximately 1,200 nucleotides in length. In the processing of the mRNA precursor, there appears to be a loss of the majority of the repetitive sequence at the 5' end. The genome contains interspersed sequences of poly (dT)25. These sequences, which appear to be at the 3' terminus of the transcription unit, are transcribed directly into the heterogenous nuclear RNA and are contained within the messenger RNA. During the processing of the heterogeneous nuclear RNA, a poly (A) sequence of approximately 125 nucleotides in length is added posttranscriptionally to the 3' end of the molecule.

Base Sequence↗

PubMed1979

Properties of a small transcribed poly A sequence in heterogeneous nuclear RNA of HeLa cells.

A class of heterogeneous nuclear RNA (hnRNA) molecules contain an internal transcribed poly A sequence of close to 25 uninterrupted AMP residues. HnRNA molecules containing this sequence are separable from those containing the large 3' terminal poly A sequence on the basis of their differential affinity for oligo dT cellulose. The fact that the transcribed small poly A and the 3' terminal poly A are not found in the same hnRNA molecules even though both are present in similar size classes and that the small poly A is absent from cytoplasmic messenger RNA (mRNA) has led us to propose a scheme for mRNA processing in which the 3' end of the small poly A in hnRNA becomes a priming size for the post-transcriptional addiction of the large poly A.

HeLa Cells↗

PubMed1975

Metabolism of the polyadenylate sequence of nuclear RNA and messenger RNA in mammalian cells.

The poly(A) sequences at the 3' end of mRNA and nuclear RNA molecules of mouse sarcoma and Chinese hamster cells are subject to an elongation process distinct from de novo synthesis. This process continues in cells treated with a high level of actinomycin D to block transcription. This results in the labeling of the steady-state poly(A) population in the cytoplasm and of unusually long poly(A) segments in the nucleus. In cells incubated with 3H-adenosine in the absence of drug treatment, cytoplasmic steady-state poly(A) segments with short labeled sequences at the 3' end can be detected by their heterogenous size distribution and by measurements of adenosine and AMP released by alkaline hydrolysis. These measurements indicate an average size of 8 residues for the labeled sequences. In the nucleus, a slow elongation of preexisting poly(A) chains can also be detected through measurements of AMP and adenosine. The cytoplasmic elongation process leads to turnover of the 3' end of the poly(A) sequence on mRNA, because of concomitant removal of AMP residues. It is apparently not linked to mRNA translation. The Chinese hamster and mouse sarcoma cells appear to differ markedly with respect to relative extents of poly(A) chain extension and de novo synthesis.

Adenine Nucleotides↗