RESUMO
Long non-coding RNAs (lncRNAs) are implicated in a plethora of cellular processes, but an in-depth understanding of their functional features or their mechanisms of action is currently lacking. Here we study Meteor, a lncRNA transcribed near the gene encoding EOMES, a pleiotropic transcription factor implicated in various processes throughout development and in adult tissues. Using a wide array of perturbation techniques, we show that transcription elongation through the Meteor locus is required for Eomes activation in mouse embryonic stem cells, with Meteor repression linked to a change in the subpopulation primed to differentiate to the mesoderm lineage. We further demonstrate that a distinct functional feature of the locus-namely, the underlying DNA element-is required for suppressing Eomes expression following neuronal differentiation. Our results demonstrate the complex regulation that can be conferred by a single locus and emphasize the importance of careful selection of perturbation techniques when studying lncRNA loci.
Assuntos
RNA Longo não Codificante , Proteínas com Domínio T , Animais , Camundongos , Diferenciação Celular/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Removal of introns during pre-mRNA splicing, which is central to gene expression, initiates by base pairing of U1 snRNA with a 5' splice site (5'SS). In mammals, many introns contain weak 5'SSs that are not efficiently recognized by the canonical U1 snRNP, suggesting alternative mechanisms exist. Here, we develop a cross-linking immunoprecipitation coupled to a high-throughput sequencing method, BCLIP-seq, to identify NRDE2 (nuclear RNAi-defective 2), and CCDC174 (coiled-coil domain-containing 174) as novel RNA-binding proteins in mouse ES cells that associate with U1 snRNA and 5'SSs. Both proteins bind directly to U1 snRNA independently of canonical U1 snRNP-specific proteins, and they are required for the selection and effective processing of weak 5'SSs. Our results reveal that mammalian cells use noncanonical splicing factors bound directly to U1 snRNA to effectively select suboptimal 5'SS sequences in hundreds of genes, promoting proper splice site choice, and accurate pre-mRNA splicing.
Assuntos
Precursores de RNA , Sítios de Splice de RNA , Animais , Camundongos , Sítios de Splice de RNA/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Interferência de RNA , Splicing de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Processamento Alternativo , Mamíferos/genéticaRESUMO
PURPOSE: The ability of next-generation sequencing (NGS) assays to interrogate thousands of genomic loci has revolutionized genetic testing. However, translation to the clinic is impeded by false-negative results that pose a risk to patients. In response, regulatory bodies are calling for reliability measures to be reported alongside NGS results. Existing methods to estimate reliability do not account for sample- and position-specific variability, which can be significant. Here, we report an approach that computes reliability metrics for every genomic position and sample interrogated by an NGS assay. METHODS: Our approach predicts the limit of detection (LOD), the lowest reliably detectable variant fraction, by taking technical factors into account. We initially explored how LOD is affected by input material amount, library conversion rate, sequencing coverage, and sequencing error rate. This revealed that LOD depends heavily on genomic context and sample properties. Using these insights, we developed a computational approach to predict LOD on the basis of a biophysical model of the NGS workflow. We focused on targeted assays for cell-free DNA, but, in principle, this approach applies to any NGS assay. RESULTS: We validated our approach by showing that it accurately predicts LOD and distinguishes reliable from unreliable results when screening 580 lung cancer samples for actionable mutations. Compared with a standard variant calling workflow, our approach avoided most false negatives and improved interassay concordance from 94% to 99%. CONCLUSION: Our approach, which we name LAVA (LOD-aware variant analysis), reports the LOD for every position and sample interrogated by an NGS assay. This enables reliable results to be identified and improves the transparency and safety of genetic tests.
Assuntos
Neoplasias Pulmonares , Nucleotídeos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Reprodutibilidade dos TestesRESUMO
Some long non-coding RNA (lncRNA) genes encode a functional RNA product, whereas others act as DNA elements or via the act of transcription . We describe here a ribozyme-based approach to deplete an endogenous lncRNA in mouse embryonic stem cells, with minimal disruption of its gene. This enables the role of the lncRNA product to be tested.
Assuntos
Células-Tronco Embrionárias/metabolismo , Edição de Genes/métodos , RNA Catalítico/genética , RNA Longo não Codificante/genética , RNA Viral/genética , Animais , Sistemas CRISPR-Cas , Células-Tronco Embrionárias/enzimologia , Camundongos , Conformação de Ácido Nucleico , RNA Catalítico/metabolismo , RNA Longo não Codificante/metabolismo , Recombinação GenéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
RNA decay is crucial for mRNA turnover and surveillance and misregulated in many diseases. This complex system is challenging to study, particularly in mammals, where it remains unclear whether decay pathways perform specialized versus redundant roles. Cytoplasmic pathways and links to translation are particularly enigmatic. By directly profiling decay factor targets and normal versus aberrant translation in mouse embryonic stem cells (mESCs), we uncovered extensive decay pathway specialization and crosstalk with translation. XRN1 (5'-3') mediates cytoplasmic bulk mRNA turnover whereas SKIV2L (3'-5') is universally recruited by ribosomes, tackling aberrant translation and sometimes modulating mRNA abundance. Further exploring translation surveillance revealed AVEN and FOCAD as SKIV2L interactors. AVEN prevents ribosome stalls at structured regions, which otherwise require SKIV2L for clearance. This pathway is crucial for histone translation, upstream open reading frame (uORF) regulation, and counteracting ribosome arrest on small ORFs. In summary, we uncovered key targets, components, and functions of mammalian RNA decay pathways and extensive coupling to translation.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Ligação a DNA/fisiologia , Exorribonucleases/fisiologia , Células-Tronco Embrionárias Murinas/metabolismo , Biossíntese de Proteínas , RNA Helicases/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fases de Leitura Aberta , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.
Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Proteínas de Ligação a RNA/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Células-Tronco Embrionárias , Humanos , Camundongos , Camundongos Knockout , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Domínios Proteicos/genética , Interferência de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms.
RESUMO
While the activity of multiprotein complexes is crucial for cellular metabolism, little is known about the mechanisms that collectively control the expression of their components. Here, we investigate the regulations targeting the biogenesis of the nuclear pore complex (NPC), the macromolecular assembly mediating nucleocytoplasmic exchanges. Systematic analysis of RNA-binding proteins interactomes, together with in vivo and in vitro assays, reveal that a subset of NPC mRNAs are specifically bound by Hek2, a yeast hnRNP K-like protein. Hek2-dependent translational repression and protein turnover are further shown to finely tune the levels of NPC subunits. Strikingly, mutations or physiological perturbations altering pore integrity decrease the levels of the NPC-associated SUMO protease Ulp1, and trigger the accumulation of sumoylated versions of Hek2 unable to bind NPC mRNAs. Our results support the existence of a quality control mechanism involving Ulp1 as a sensor of NPC integrity and Hek2 as a repressor of NPC biogenesis.
Assuntos
Cisteína Endopeptidases/metabolismo , Retroalimentação Fisiológica , Poro Nuclear/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Biologia Computacional , Conjuntos de Dados como Assunto , Ligação Proteica/fisiologia , RNA Mensageiro/metabolismo , Sumoilação/fisiologiaRESUMO
Numerous links exist between co-transcriptional RNA processing and the transcribing RNAPII. In particular, pre-mRNA splicing was reported to be associated with slowed RNAPII elongation. Here, we identify a site of ubiquitination (K1246) in the catalytic subunit of RNAPII close to the DNA entry path. Ubiquitination was increased in the absence of the Bre5-Ubp3 ubiquitin protease complex. Bre5 binds RNA in vivo, with a preference for exon 2 regions of intron-containing pre-mRNAs and poly(A) proximal sites. Ubiquitinated RNAPII showed similar enrichment. The absence of Bre5 led to impaired splicing and defects in RNAPII elongation in vivo on a splicing reporter construct. Strains expressing RNAPII with a K1246R mutation showed reduced co-transcriptional splicing. We propose that ubiquinitation of RNAPII is induced by RNA processing events and linked to transcriptional pausing, which is released by Bre5-Ubp3 associated with the nascent transcript.
Assuntos
Domínio Catalítico , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Ubiquitinação , Endopeptidases/metabolismo , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprogramming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV crosslinking immediately following glucose withdrawal (0, 4, and 8 min). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many growth-related mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. We conclude that transcription termination is followed by TRAMP-mediated RNA decay. Upregulated transcripts evaded increased surveillance factor binding following glucose withdrawal. Some upregulated genes showed use of alternative transcription starts to bypass strong NNS binding sites. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability.
Assuntos
Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Adaptação Fisiológica , Sítios de Ligação , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Glucose/deficiência , Glucose/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Nuclear/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
Reversible modification of the RNAPII C-terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand-specific, nucleotide-resolution information, and we used a machine learning-based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3', with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3' ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein-coding genes and persisted throughout exon 1 of intron-containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein-coding genes.
Assuntos
RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Sítios de Ligação , Aprendizado de Máquina , Cadeias de Markov , Fosforilação , RNA Polimerase II/química , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.
Assuntos
Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Terminação da Transcrição Genética , Regiões 3' não Traduzidas , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Embryonic stem cell (ESC) culture conditions are important for maintaining long-term self-renewal, and they influence cellular pluripotency state. Here, we report single cell RNA-sequencing of mESCs cultured in three different conditions: serum, 2i, and the alternative ground state a2i. We find that the cellular transcriptomes of cells grown in these conditions are distinct, with 2i being the most similar to blastocyst cells and including a subpopulation resembling the two-cell embryo state. Overall levels of intercellular gene expression heterogeneity are comparable across the three conditions. However, this masks variable expression of pluripotency genes in serum cells and homogeneous expression in 2i and a2i cells. Additionally, genes related to the cell cycle are more variably expressed in the 2i and a2i conditions. Mining of our dataset for correlations in gene expression allowed us to identify additional components of the pluripotency network, including Ptma and Zfp640, illustrating its value as a resource for future discovery.
Assuntos
Células-Tronco Embrionárias Murinas/fisiologia , RNA/genética , Transcriptoma , Animais , Diferenciação Celular/genética , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Célula ÚnicaRESUMO
The RNA component of signal recognition particle (SRP) is transcribed by RNA polymerase III, and most steps in SRP biogenesis occur in the nucleolus. Here, we examine processing and quality control of the yeast SRP RNA (scR1). In common with other pol III transcripts, scR1 terminates in a U-tract, and mature scR1 retains a U4-5 sequence at its 3' end. In cells lacking the exonuclease Rex1, scR1 terminates in a longer U5-6 tail that presumably represents the primary transcript. The 3' U-tract of scR1 is protected from aberrant processing by the La homologue, Lhp1 and overexpressed Lhp1 apparently competes with both the RNA surveillance system and SRP assembly factors. Unexpectedly, the TRAMP and exosome nuclear RNA surveillance complexes are also implicated in protecting the 3' end of scR1, which accumulates in the nucleolus of cells lacking the activities of these complexes. Misassembled scR1 has a primary degradation pathway in which Rrp6 acts early, followed by TRAMP-stimulated exonuclease degradation by the exosome. We conclude that the RNA surveillance machinery has key roles in both SRP biogenesis and quality control of the RNA, potentially facilitating the decision between these alternative fates.
Assuntos
Núcleo Celular/metabolismo , Processamento de Terminações 3' de RNA , RNA Fúngico/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Nucléolo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Estabilidade de RNA , RNA Fúngico/química , RNA Citoplasmático Pequeno/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic genomes generate a heterogeneous ensemble of mRNAs and long noncoding RNAs (lncRNAs). LncRNAs and mRNAs are both transcribed by Pol II and acquire 5' caps and poly(A) tails, but only mRNAs are translated into proteins. To address how these classes are distinguished, we identified the transcriptome-wide targets of 13 RNA processing, export, and turnover factors in budding yeast. Comparing the maturation pathways of mRNAs and lncRNAs revealed that transcript fate is largely determined during 3' end formation. Most lncRNAs are targeted for nuclear RNA surveillance, but a subset with 3' cleavage and polyadenylation features resembling the mRNA consensus can be exported to the cytoplasm. The Hrp1 and Nab2 proteins act at this decision point, with dual roles in mRNA cleavage/polyadenylation and lncRNA surveillance. Our data also reveal the dynamic and heterogeneous nature of mRNA maturation, and highlight a subset of "lncRNA-like" mRNAs regulated by the nuclear surveillance machinery.
Assuntos
RNA Fúngico/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcriptoma , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Longo não Codificante/química , RNA Mensageiro/química , Ribonucleoproteínas/química , Saccharomyces cerevisiae/genéticaRESUMO
Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3' end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA-protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA-18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3' guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA-protein complex are potentially amenable to this approach.
Assuntos
Ribossomos/química , Ribossomos/metabolismo , Sequência de Bases , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ésteres do Ácido SulfúricoRESUMO
The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. Here we apply in vivo RNA crosslinking (CRAC) to the nucleases (Rrp44, Rrp6), two structural subunits (Rrp41, Csl4) and a cofactor (Trf4) of the yeast exosome. Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of non-coding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome indicating that substrates follow multiple pathways to the nucleases.
Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/genética , Perfilação da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , Northern Blotting , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Regulação Fúngica da Expressão Gênica , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In this issue of Molecular Cell, single-cell analyses by Bumgarner et al. (2012) reveal how two antagonistic long noncoding RNAs at the FLO11 locus define a toggle responsible for morphological heterogeneity in genetically identical populations of budding yeast.