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Results for “RNA 3' End Processing”

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PubMed1994

3' end processing of mouse histone pre-mRNA: evidence for additional base-pairing between U7 snRNA and pre-mRNA.

We have analysed the extent of base-pairing interactions between spacer sequences of histone pre-mRNA and U7 snRNA present in the trans-acting U7 snRNP and their importance for histone RNA 3' end processing in vitro. For the efficiently processed mouse H4-12 gene, a computer analysis revealed that additional base pairs could be formed with U7 RNA outside of the previously recognised spacer element (stem II). One complementarity (stem III) is located more 3' and involves nucleotides from the very 5' end of U7 RNA. The other, more 5' located complementarity (stem I) involves nucleotides of the Sm binding site of U7 RNA, a part known to interact with snRNP structural proteins. These potential stem structures are separated from each other by short internal loops of unpaired nucleotides. Mutational analyses of the pre-mRNA indicate that stems II and III are equally important for interaction with the U7 snRNP and for processing, whereas mutations in stem I have moderate effects on processing efficiency, but do not impair complex formation with the U7 snRNP. Thus nucleotides near the processing site may be important for processing, but do not contribute to the assembly of an active complex by forming a stem I structure. The importance of stem III was confirmed by the ability of a complementary mutation in U7 RNA to suppress a stem III mutation in a complementation assay using Xenopus laevis oocytes. The main role of the factor(s) binding to the upstream hairpin loop is to stabilise the U7-pre-mRNA complex. This was shown by either stabilising (by mutation) or destabilising (by increased temperature) the U7-pre-mRNA base-pairing under conditions where hairpin factor binding was either allowed or prevented (by mutation or competition). The hairpin dependence of processing was found to be inversely related to the strength of the U7-pre-mRNA interaction.

Animals↗

PubMed2003

A 10 residue motif at the C-terminus of the RNA pol II CTD is required for transcription, splicing and 3' end processing.

The RNA polymerase II C-terminal heptad repeat domain (CTD) is essential for normal transcription and co-transcriptional processing of mRNA precursors. The mammalian CTD comprises 52 heptads whose consensus, YSPTSPS, is conserved throughout eukaryotes, followed by a 10 amino acid C-terminal sequence that is conserved only among vertebrates. Here we show that surprisingly, the heptad repeats are not sufficient to support efficient transcription, pre-mRNA processing or full cell viability. In addition to the heptads, the 10 amino acid C-terminal motif is essential for high level transcription, splicing and poly(A) site cleavage. Efficient mRNA synthesis from a transiently transfected reporter gene required the C-terminal motif plus between 16 and 25 heptad repeats from either the N- or C-terminal half of the CTD. Twenty-seven consensus heptads plus the C-terminal motif also supported efficient mRNA synthesis but not cell viability.

Amino Acid Motifs↗

PubMed2004

The link between mRNA processing and transcription: communication works both ways.

Many pre-mRNA processing events including 5' end capping, splicing out introns, and 3' end maturation by cleavage or polyadenylation occur while the nascent RNA chain is being synthesized by RNA polymerase II. As a consequence of this arrangement, the physiological substrate for most processing factors is not a solitary pre-RNA but instead a ternary complex comprising a growing RNA chain spewing from the exit channel of an RNA polymerase II molecule as it speeds along a chromatin template at 1000-2000 bases/min. mRNA processing factors make protein-protein contacts with elongating pol II in a complex we have dubbed the "mRNA factory," which carries out synthesis, processing, and packaging of the transcript. Recent studies have shown that the "mRNA factory" is a dynamic complex whose composition changes as it traverses the length of a gene. This complex is also the setting for a growing number of regulatory interactions, which influence the function of both the processing and transcription machineries.

Animals↗

PubMed2005

P-TEFb is not an essential elongation factor for the intronless human U2 snRNA and histone H2b genes.

Phosphorylation of Ser2 of the heptapeptide repeat of the CTD of mammalian pol II by P-TEFb is associated with productive elongation of transcription of protein-coding genes. Here, we show that the CTD of pol II transcribing the human U2 snRNA genes is phosphorylated on Ser2 in vivo and that both the CDK9 kinase and cyclin T components of P-TEFb are required for cotranscriptional recognition of the 3' box RNA 3' end processing signal. However, inhibitors of CDK9 do not affect transcription of the U2 genes, indicating that P-TEFb functions exclusively as an RNA processing factor in expression of these relatively short, intronless genes. We also show that inhibition of CDK9 does not adversely affect either transcription of an intron-less, replication-activated histone H2b gene or recognition of the histone gene-specific U7-dependent RNA 3' end formation signal. These results emphasize that the role of P-TEFb as an activator of transcription elongation can be separated from its role in RNA processing and that neither function is universally required for expression of mammalian pol II-dependent genes.

Amino Acid Sequence↗

PubMed2002

Intron status and 3'-end formation control cotranscriptional export of mRNA.

Messenger RNA export factors are recruited to genes in a transcription-dependent manner. To ascertain the mechanism of this process, we show that RNA polymerase II transcription is sufficient to recruit the Saccharomyces cerevisiae hnRNP protein Npl3 to a gene independent of RNA sequence. In contrast, the cotranscriptional recruitment of the RNA-binding protein Yra1 is dependent on pre-mRNA processing. Yra1 associates with introns of intron-containing genes in a splicing-dependent manner. Conversely, Yra1 recruitment to genes without introns is not dependent on splicing. Finally, 3'-end formation is required for Yra1 recruitment to genes regardless of intron status.

Heterogeneous-Nuclear Ribonucleoproteins↗

PubMed2005

Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 3'-extension of 5S rRNA in Saccharomyces cerevisiae.

Rrs1p, a ribosomal protein L11-binding protein, has an essential role in biogenesis of 60S ribosomal subunits. We obtained conditionally synthetic lethal allele with the rrs1-5 mutation and determined that the mutation is in REX1, which encodes an exonuclease. The highly conserved leucine at 305 was substituted with tryptophan in rex1-1. The rex1-1 allele resulted in 3'-extended 5S rRNA. Polysome analysis revealed that rex1-1 and rrs1-5 caused a synergistic defect in the assembly of 60S ribosomal subunits. In vivo and in vitro binding assays indicate that Rrs1p interacts with the ribosomal protein L5-5S rRNA complex. The rrs1-5 mutation weakens the interaction between Rrs1p with both L5 and L11. These data suggest that the assembly of L5-5S rRNA on 60S ribosomal subunits coordinates with assembly of L11 via Rrs1p.

Cell Growth Processes↗

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↗

PubMed2004

New perspectives on connecting messenger RNA 3' end formation to transcription.

Recent advances in understanding the molecular mechanism of mRNA 3' end cleavage and polyadenylation have uncovered an unanticipated involvement of this process in the regulation of the transcriptional apparatus on its chromatin template. Thus, newly defined factors associated with mRNA 3' end formation are also connected with initiation of transcription, suggesting a close collaboration between the initiation and termination phases of transcription. Furthermore several of these factors are involved in setting up appropriate chromatin structure to facilitate efficient transcriptional elongation and termination.

Animals↗

PubMed2005

From transcription to mRNA: PAF provides a new path.

Two new studies reveal a novel connection between transcription and pre-mRNA processing: the Paf1 elongation complex is involved in the 3' end formation of polyadenylated and nonpolyadenylated RNA polymerase II transcripts.

Gene Expression Regulation↗

PubMed2002

Structure of the histone mRNA hairpin required for cell cycle regulation of histone gene expression.

Expression of replication-dependent histone genes requires a conserved hairpin RNA element in the 3' untranslated regions of poly(A)-less histone mRNAs. The 3' hairpin element is recognized by the hairpin-binding protein or stem-loop-binding protein (HBP/SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage generating the mature mRNA 3' end. The 3' hairpin and presumably HBP/SLBP are also required for nucleocytoplasmic transport, translation, and stability of histone mRNAs. RNA 3' processing and mRNA stability are both regulated during the cell cycle. Here, we have determined the three-dimensional structure of a 24-mer RNA comprising a mammalian histone RNA hairpin using heteronuclear multidimensional NMR spectroscopy. The hairpin adopts a novel UUUC tetraloop conformation that is stabilized by base stacking involving the first and third loop uridines and a closing U-A base pair, and by hydrogen bonding between the first and third uridines in the tetraloop. The HBP interaction of hairpin RNA variants was analyzed in band shift experiments. Particularly important interactions for HBP recognition are mediated by the closing U-A base pair and the first and third loop uridines, whose Watson-Crick functional groups are exposed towards the major groove of the RNA hairpin. The results obtained provide novel structural insight into the interaction of the histone 3' hairpin with HBP, and thus the regulation of histone mRNA metabolism.

Animals↗

PubMed2005

Connections between mRNA 3' end processing and transcription termination.

Discoveries within the last few years have revealed that the multiple steps in gene expression are remarkably integrated. There have recently been several advances in deciphering how mRNA 3' end processing is linked with transcription elongation and termination. It has been known for quite a long time that transcription termination is somehow intertwined with polyadenylation, but it is still unclear exactly how these two processes influence each other. Some recent reports are shedding light on these connections.

Animals↗

PubMed2005

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Alleles↗

PubMed2005

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana.

Arabidopsis thaliana chloroplasts contain at least two 3' to 5' exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3' processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5' and 3' ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3' extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.

Arabidopsis↗

PubMed1995

The effects of differential polyadenylation on expression of the dihydrofolate reductase-encoding gene in Chinese hamster lung cells.

Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR and mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3' end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+ mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3' end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression.

Alleles↗

PubMed1990

Proximity to the promoter inhibits recognition of cauliflower mosaic virus polyadenylation signal.

The replication of retroviruses and pararetroviruses (such as caulimo- and hepadnaviruses) involves the production of terminally redundant genomic-length RNA. The sequence repeated at both ends of the RNA (the R region) contains a polyadenylation signal, and for production of full-length RNA the version of this at the 5' end of the template must be bypassed by RNA polymerase, but the version at the 3' end must be recognized. This implies that the position of the polyadenylation signal determines its efficiency, and we report here experiments aimed at investigating the basis of this phenomenon. Our results with cauliflower mosaic virus suggest that proximity to the transcription initiation site inhibits messenger RNA 3'-end processing directed by polyadenylation signals.

DNA, Recombinant↗

PubMed2004

Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo.

Although correct tRNA 3' ends are crucial for protein biosynthesis, generation of mature tRNA 3' ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3' end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3' endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3' end processing. In addition our data show that in vivo 5' processing of nuclear and mitochondrial pre-tRNAs occurs before 3' processing.

Amino Acid Sequence↗

PubMed2002

Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry.

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.

Chromatography, Affinity↗

PubMed1997

Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation.

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

Adenoviruses, Human↗