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1.
Nature ; 583(7816): 361-362, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32669690

Assuntos
Catenanos , Poli A
2.
PLoS One ; 15(6): e0234696, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32544193

RESUMO

Poly(A) tails at the 3' end of eukaryotic messenger RNAs control mRNA stability and translation efficiency. Facilitated by various NGS methods, alternative polyadenylation sites determining the 3'-UTR length of gene transcripts have been extensively studied. However, poly(A) lengths demonstrating dynamic and developmental regulation remain largely unexplored. The recently developed NGS-based methods for genome-wide poly(A) profiling have promoted the study of genom-wide poly(A) dynamics. Here we present a straight forward NGS-method for poly(A) profiling, which applies a direct 3'-end adaptor ligation and the template switching for 5'-end adaptor ligation for cDNA library construction. Poly(A) lengths are directly calculated from base call data using a self-developed pipeline pA-finder. The libraries were directly sequenced from the 3'-UTR regions into the followed poly(A) tails, firstly on NextSeq 500 to produce single-end 300-nt reads, demonstrating the method feasibility and that optimization of the fragmented RNA size for cDNA library construction could detecting longer poly (A) tails. We next applied Poly(A)-seq cDNA libraries containing 40-nt and 120-nt poly(A) tail spike-in RNAs on HiSeq X-ten and NovaSeq 6000 to obtain 150-nt and 250-nt pair-end reads. The sequencing profiles of the spike-in RNAs demonstrated both high accuracy and high quality score in reading poly(A) tails. The poly(A) signal bleeding into the 3' adaptor sequence and a sharp decreased quality score at the junction were observed, allowing the modification of pA-finder to remove homopolymeric signal bleeding. We hope that wide applications of Poly(A)-seq help facilitate the study of the development- and disease-related poly(A) dynamics and regulation, and of the recent emerging mixed tailing regulation.


Assuntos
Poli A/genética , Análise de Sequência de RNA/métodos , Regiões 3' não Traduzidas/genética , Biblioteca Gênica , Genoma , Humanos , Poliadenilação/genética , Transcriptoma/genética
4.
Nucleic Acids Res ; 48(11): 5926-5938, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421815

RESUMO

Alternative polyadenylation (APA) produces isoforms with distinct 3'-ends, yet their functional differences remain largely unknown. Here, we introduce the APA-seq method to detect the expression levels of APA isoforms from 3'-end RNA-Seq data by exploiting both paired-end reads for gene isoform identification and quantification. We detected the expression levels of APA isoforms in individual Caenorhabditis elegans embryos at different stages throughout embryogenesis. Examining the correlation between the temporal profiles of isoforms led us to distinguish two classes of genes: those with highly correlated isoforms (HCI) and those with lowly correlated isoforms (LCI) across time. We hypothesized that variants with similar expression profiles may be the product of biological noise, while the LCI variants may be under tighter selection and consequently their distinct 3' UTR isoforms are more likely to have functional consequences. Supporting this notion, we found that LCI genes have significantly more miRNA binding sites, more correlated expression profiles with those of their targeting miRNAs and a relative lack of correspondence between their transcription and protein abundances. Collectively, our results suggest that a lack of coherence among the regulation of 3' UTR isoforms is a proxy for selective pressures acting upon APA usage and consequently for their functional relevance.


Assuntos
Regiões 3' não Traduzidas/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Poli A/análise , Poliadenilação , Animais , Drosophila melanogaster , Desenvolvimento Embrionário/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Xenopus laevis
5.
PLoS Comput Biol ; 16(4): e1007842, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339166

RESUMO

The mammalian circadian clock is deeply rooted in rhythmic regulation of gene expression. Rhythmic transcriptional control mediated by the circadian transcription factors is thought to be the main driver of mammalian circadian gene expression. However, mounting evidence has demonstrated the importance of rhythmic post-transcriptional controls, and it remains unclear how the transcriptional and post-transcriptional mechanisms collectively control rhythmic gene expression. In mouse liver, hundreds of genes were found to exhibit rhythmicity in poly(A) tail length, and the poly(A) rhythms are strongly correlated with the protein expression rhythms. To understand the role of rhythmic poly(A) regulation in circadian gene expression, we constructed a parsimonious model that depicts rhythmic control imposed upon basic mRNA expression and poly(A) regulation processes, including transcription, deadenylation, polyadenylation, and degradation. The model results reveal the rhythmicity in deadenylation as the strongest contributor to the rhythmicity in poly(A) tail length and the rhythmicity in the abundance of the mRNA subpopulation with long poly(A) tails (a rough proxy for mRNA translatability). In line with this finding, the model further shows that the experimentally observed distinct peak phases in the expression of deadenylases, regardless of other rhythmic controls, can robustly cluster the rhythmic mRNAs by their peak phases in poly(A) tail length and abundance of the long-tailed subpopulation. This provides a potential mechanism to synchronize the phases of target gene expression regulated by the same deadenylases. Our findings highlight the critical role of rhythmic deadenylation in regulating poly(A) rhythms and circadian gene expression.


Assuntos
Relógios Circadianos/genética , Regulação da Expressão Gênica/genética , Poli A , Poliadenilação/genética , Adenina/metabolismo , Animais , Biologia Computacional , Simulação por Computador , Fígado/metabolismo , Camundongos , Modelos Genéticos , Poli A/genética , Poli A/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Nucleic Acids Res ; 48(10): 5349-5365, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313933

RESUMO

Growing mammalian oocytes accumulate substantial amounts of RNA, most of which is degraded during subsequent meiotic maturation. The growth-to-maturation transition begins with germinal vesicle or nuclear envelope breakdown (GVBD) and is critical for oocyte quality and early development. The molecular machinery responsible for the oocyte transcriptome transition remains unclear. Here, we report that an exosome-associated RNase, EXOSC10, sculpts the transcriptome to facilitate the growth-to-maturation transition of mouse oocytes. We establish an oocyte-specific conditional knockout of Exosc10 in mice using CRISPR/Cas9 which results in female subfertility due to delayed GVBD. By performing multiple single oocyte RNA-seq, we document dysregulation of several types of RNA, and the mRNAs that encode proteins important for endomembrane trafficking and meiotic cell cycle. As expected, EXOSC10-depleted oocytes have impaired endomembrane components including endosomes, lysosomes, endoplasmic reticulum and Golgi. In addition, CDK1 fails to activate, possibly due to persistent WEE1 activity, which blocks lamina phosphorylation and disassembly. Moreover, we identified rRNA processing defects that cause higher percentage of developmentally incompetent oocytes after EXOSC10 depletion. Collectively, we propose that EXOSC10 promotes normal growth-to-maturation transition in mouse oocytes by sculpting the transcriptome to degrade RNAs encoding growth-phase factors and, thus, support the maturation phase of oogenesis.


Assuntos
Exorribonucleases/fisiologia , Complexo Multienzimático de Ribonucleases do Exossomo/fisiologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese , Transcriptoma , Animais , Proteína Quinase CDC2/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Feminino , Infertilidade Feminina/genética , Membranas Intracelulares/metabolismo , Camundongos , Lâmina Nuclear/metabolismo , Poli A , RNA/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , RNA-Seq
7.
Nucleic Acids Res ; 48(10): 5766-5776, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32313953

RESUMO

Aberrant KRAS signaling is a driver of many cancers and yet remains an elusive target for drug therapy. The nuclease hypersensitive element of the KRAS promoter has been reported to form secondary DNA structures called G-quadruplexes (G4s) which may play important roles in regulating KRAS expression, and has spurred interest in structural elucidation studies of the KRAS G-quadruplexes. Here, we report the first high-resolution crystal structure (1.6 Å) of a KRAS G-quadruplex as a 5'-head-to-head dimer with extensive poly-A π-stacking interactions observed across the dimer. Molecular dynamics simulations confirmed that the poly-A π-stacking interactions are also maintained in the G4 monomers. Docking and molecular dynamics simulations with two G4 ligands that display high stabilization of the KRAS G4 indicated the poly-A loop was a binding site for these ligands in addition to the 5'-G-tetrad. Given sequence and structural variability in the loop regions provide the opportunity for small-molecule targeting of specific G4s, we envisage this high-resolution crystal structure for the KRAS G-quadruplex will aid in the rational design of ligands to selectively target KRAS.


Assuntos
Quadruplex G , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Cristalografia por Raios X , DNA/química , Dimerização , Ligantes , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Água/química
8.
Mol Cell ; 78(3): 434-444.e5, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32294471

RESUMO

Gene expression is regulated by the rates of synthesis and degradation of mRNAs, but how these processes are coordinated is poorly understood. Here, we show that reduced transcription dynamics of specific genes leads to enhanced m6A deposition, preferential activity of the CCR4-Not complex, shortened poly(A) tails, and reduced stability of the respective mRNAs. These effects are also exerted by internal ribosome entry site (IRES) elements, which we found to be transcriptional pause sites. However, when transcription dynamics, and subsequently poly(A) tails, are globally altered, cells buffer mRNA levels by adjusting the expression of mRNA degradation machinery. Stress-provoked global impediment of transcription elongation leads to a dramatic inhibition of the mRNA degradation machinery and massive mRNA stabilization. Accordingly, globally enhanced transcription, such as following B cell activation or glucose stimulation, has the opposite effects. This study uncovers two molecular pathways that maintain balanced gene expression in mammalian cells by linking transcription to mRNA stability.


Assuntos
Poli A/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Adenosina/análogos & derivados , Animais , Linfócitos B/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Sítios Internos de Entrada Ribossomal , Células MCF-7 , Camundongos Endogâmicos C57BL , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Poli A/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , Receptores CCR4/genética , Receptores CCR4/metabolismo
9.
RNA ; 26(5): 613-628, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32111664

RESUMO

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'-3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.


Assuntos
Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA/genética , Ribose/química , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Células Eucarióticas , Fases de Leitura Aberta/genética , Poli A/genética , Biossíntese de Proteínas/genética , Capuzes de RNA/genética , Ribose/genética , Ribosemonofosfatos/química , Ribosemonofosfatos/genética
10.
Nucleic Acids Res ; 48(5): 2564-2578, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31993626

RESUMO

Ribonuclease P (RNase P) is essential for the 5'-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5'-immature tRNAs can be aminoacylated unless their 5'-extensions ≥8 nt. Surprisingly, the failure of 5'-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5'-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Óperon/genética , Precursores de RNA/biossíntese , Ribonuclease P/metabolismo , Aminoacilação , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Mutação/genética , Poli A/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Nucleic Acids Res ; 48(5): 2518-2530, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31950173

RESUMO

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exossomos/metabolismo , Poli A/metabolismo , Estabilidade de RNA , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Proteoma/metabolismo , Ribonucleoproteínas/metabolismo
13.
EMBO J ; 39(3): e103365, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31858614

RESUMO

Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Códon , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Poli A/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética
14.
Mikrochim Acta ; 187(1): 20, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31807965

RESUMO

A multi-channel localized surface plasmon resonance system is described for absorptiometric determination of abscisic acid (ABA). The system is making use of gold nanoparticles and consists of a broadband light source, a multi-channel alignment device, and a fiber spectrometer. The method is based on the specific interaction between an ABA-binding aptamer and ABA. This induces the growth of gold nanoparticles (AuNPs) functionalized with a polyadenine-tailed aptamer that act as optical probes. Different concentrations of ABA give rise to varied morphologies of grown AuNPs. This causes a change of absorption spectra which is recorded by the system. ABA can be quantified by measurement of the peak wavelength shifts of grown AuNPs. Under optimized conditions, this method shows a linear relationship in the 1 nM to 10 µM ABA concentration range. The detection limit is 0.51 nM. The sensitivity of the ABA assay is strongly improved compared to the method based on salt-induced AuNP aggregation. This is attributed to the use of a poly-A-tailed aptamer and the catalytic ability of AuNPs. In the actual application, the ABA concentration of ABA in fresh leaves of rice is measured with the maximum relative error of 8.03% in comparison with the ELISA method. Graphical abstractSchematic representation of an absorptiometric approach for determination of abscisic acid based on the growth of polyA-tailed aptamer-AuNPs probes and a multi-channel localized surface plasmon resonance system.


Assuntos
Ácido Abscísico/análise , Aptâmeros de Nucleotídeos/química , Ouro/química , Nanopartículas Metálicas/química , Poli A/química , Ressonância de Plasmônio de Superfície/métodos , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Absorção Fisico-Química , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Oryza/química
15.
Int J Mol Sci ; 21(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31877939

RESUMO

The DNA in living cells can be effectively damaged by high-energy radiation, which can lead to cell death. Through the ionization of water molecules, highly reactive secondary species such as low-energy electrons (LEEs) with the most probable energy around 10 eV are generated, which are able to induce DNA strand breaks via dissociative electron attachment. Absolute DNA strand break cross sections of specific DNA sequences can be efficiently determined using DNA origami nanostructures as platforms exposing the target sequences towards LEEs. In this paper, we systematically study the effect of the oligonucleotide length on the strand break cross section at various irradiation energies. The present work focuses on poly-adenine sequences (d(A4), d(A8), d(A12), d(A16), and d(A20)) irradiated with 5.0, 7.0, 8.4, and 10 eV electrons. Independent of the DNA length, the strand break cross section shows a maximum around 7.0 eV electron energy for all investigated oligonucleotides confirming that strand breakage occurs through the initial formation of negative ion resonances. When going from d(A4) to d(A16), the strand break cross section increases with oligonucleotide length, but only at 7.0 and 8.4 eV, i.e., close to the maximum of the negative ion resonance, the increase in the strand break cross section with the length is similar to the increase of an estimated geometrical cross section. For d(A20), a markedly lower DNA strand break cross section is observed for all electron energies, which is tentatively ascribed to a conformational change of the dA20 sequence. The results indicate that, although there is a general length dependence of strand break cross sections, individual nucleotides do not contribute independently of the absolute strand break cross section of the whole DNA strand. The absolute quantification of sequence specific strand breaks will help develop a more accurate molecular level understanding of radiation induced DNA damage, which can then be used for optimized risk estimates in cancer radiation therapy.


Assuntos
Dano ao DNA , DNA/química , Elétrons , Modelos Químicos , Nanoestruturas/química , Oligonucleotídeos/química , Poli A/química
16.
Nat Commun ; 10(1): 5292, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757970

RESUMO

Message RNA poly(A) tails are vital for their function and regulation. However, the full-length sequence of mRNA isoforms with their poly(A) tails remains undetermined. Here, we develop a method at single-cell level sensitivity that enables quantification of poly(A) tails along with the full-length cDNA while reading non-adenosine residues within poly(A) tails precisely, which we name poly(A) inclusive RNA isoform sequencing (PAIso-seq). Using this method, we can quantify isoform specific poly(A) tail length. More interestingly, we find that 17% of the mRNAs harbor non-A residues within the body of poly(A) tails in mouse GV oocytes. We show that PAIso-seq is sensitive enough to analyze single GV oocytes. These findings will not only provide an accurate and sensitive tool in studying poly(A) tails, but also open a door for the function and regulation of non-adenosine modifications within the body of poly(A) tails.


Assuntos
Nucleotídeos de Citosina/química , Nucleotídeos de Guanina/química , Oócitos/metabolismo , Poli A/química , RNA Mensageiro/química , Análise de Sequência de RNA/métodos , Nucleotídeos de Uracila/química , Animais , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Nucleotídeos de Citosina/análise , DNA (Citosina-5-)-Metiltransferase 1/genética , Nucleotídeos de Guanina/análise , Camundongos , Poli A/análise , Biossíntese de Proteínas , Isoformas de RNA , RNA Mensageiro/análise , Análise de Célula Única , Ativador de Plasminogênio Tecidual/genética , Nucleotídeos de Uracila/análise
17.
Nat Methods ; 16(12): 1297-1305, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740818

RESUMO

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.


Assuntos
Sequenciamento por Nanoporos/métodos , Poli A/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Células Cultivadas , Humanos
18.
Nanoscale ; 11(46): 22216-22221, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31728476

RESUMO

Poly(A-T)-rich sequences have been applied as stems of DNA dumbbell tiles for construction of single crystalline 2D DNA lattice arrays in slightly acidic solutions. These arrays show much higher stability and better organised crystalline lattice structures than those assembled from DNA dumbbell tiles with randomly sequenced stems in slightly alkaline environments. DNA nanotechnology probably provides a useful platform to study the mechanical properties of DNA duplexes with specific sequences.


Assuntos
DNA/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido Nucleico , Poli A/química , Poli T/química
19.
Mol Cell ; 76(6): 896-908.e4, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31677974

RESUMO

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/metabolismo , Proteína Fosfatase 1/metabolismo , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Terminação da Transcrição Genética , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Exorribonucleases/genética , Células HEK293 , Humanos , Cinética , Proteínas Nucleares/genética , Fosforilação , Poli A/metabolismo , Ligação Proteica , Proteína Fosfatase 1/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Fatores de Elongação da Transcrição/genética
20.
Nat Struct Mol Biol ; 26(12): 1132-1140, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768042

RESUMO

Faulty or damaged messenger RNAs are detected by the cell when translating ribosomes stall during elongation and trigger pathways of mRNA decay, nascent protein degradation and ribosome recycling. The most common mRNA defect in eukaryotes is probably inappropriate polyadenylation at near-cognate sites within the coding region. How ribosomes stall selectively when they encounter poly(A) is unclear. Here, we use biochemical and structural approaches in mammalian systems to show that poly-lysine, encoded by poly(A), favors a peptidyl-transfer RNA conformation suboptimal for peptide bond formation. This conformation partially slows elongation, permitting poly(A) mRNA in the ribosome's decoding center to adopt a ribosomal RNA-stabilized single-stranded helix. The reconfigured decoding center clashes with incoming aminoacyl-tRNA, thereby precluding elongation. Thus, coincidence detection of poly-lysine in the exit tunnel and poly(A) in the decoding center allows ribosomes to detect aberrant mRNAs selectively, stall elongation and trigger downstream quality control pathways essential for cellular homeostasis.


Assuntos
Peptídeos/metabolismo , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Peptídeos/química , Poli A/química , Poliadenilação , Polilisina/química , Polilisina/metabolismo , Estabilidade de RNA , RNA Mensageiro/química , RNA de Transferência/química , RNA de Transferência/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/química
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