RESUMO
RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3' untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Ustilago/genética , Sítios de Ligação , Transporte Biológico/genética , Endossomos/genética , Regulação da Expressão Gênica , Microtúbulos/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Transcriptoma/genéticaRESUMO
Micronuclei resulting from improper chromosome segregation foster chromosome rearrangements.1,2 To prevent micronuclei formation in mitosis, the dynamic plus ends of bundled kinetochore microtubules (k-fibers) must establish bipolar attachment with all sister kinetochores on chromosomes,3 whereas k-fiber minus ends must be clustered at the two opposing spindle poles, which are normally connected with centrosomes.4 The establishment of chromosome biorientation via k-fiber plus ends is carefully monitored by the spindle assembly checkpoint (SAC).5 However, how k-fiber minus-end clustering near centrosomes is maintained and monitored remains poorly understood. Here, we show that degradation of NuMA by auxin-inducible degron technologies results in micronuclei formation through k-fiber minus-end detachment from spindle poles during metaphase in HCT116 colon cancer cells. Importantly, k-fiber minus-end detachment from one pole creates misaligned chromosomes that maintain chromosome biorientation and satisfy the SAC, resulting in abnormal chromosome segregation. NuMA depletion also causes minus-end clustering defects in non-transformed Rpe1 cells, but it additionally induces centrosome detachment from partially focused poles, resulting in highly disorganized anaphase. Moreover, we find that NuMA depletion causes centrosome clustering defects in tetraploid-like cells, leading to an increased frequency of multipolar divisions. Together, our data indicate that NuMA is required for faithful chromosome segregation in human mitotic cells, generally by maintaining k-fiber minus-end clustering but also by promoting spindle pole-centrosome or centrosome-centrosome connection in specific cell types or contexts. Similar to erroneous merotelic kinetochore attachments,6 detachment of k-fiber minus ends from spindle poles evades spindle checkpoint surveillance and may therefore be a source of genomic instability in dividing cells.
Assuntos
Fuso Acromático , Polos do Fuso , Humanos , Centrossomo/metabolismo , Segregação de Cromossomos , Cinetocoros , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Polos do Fuso/metabolismoRESUMO
The nuclear mitotic apparatus (NuMA) protein is well conserved in vertebrates, and dynamically changes its subcellular localization from the interphase nucleus to the mitotic/meiotic spindle poles and the mitotic cell cortex. At these locations, NuMA acts as a key structural hub in nuclear formation, spindle assembly, and mitotic spindle positioning, respectively. To achieve its variable functions, NuMA interacts with multiple factors, including DNA, microtubules, the plasma membrane, importins, and cytoplasmic dynein. The binding of NuMA to dynein via its N-terminal domain drives spindle pole focusing and spindle positioning, while multiple interactions through its C-terminal region define its subcellular localizations and functions. In addition, NuMA can self-assemble into high-ordered structures which likely contribute to spindle positioning and nuclear formation. In this review, we summarize recent advances in NuMA's domains, functions and regulations, with a focus on human NuMA, to understand how and why vertebrate NuMA participates in these functions in comparison with invertebrate NuMA-related proteins.
RESUMO
Based on recent findings, long noncoding (lnc) RNAs represent a potential class of functional molecules within the cell. In this chapter we describe a computational scheme to identify and classify lncRNAs within maize from full-length cDNA sequences to designate subsets of lncRNAs for which biogenesis and regulatory mechanisms may be verified at the bench. We make use of the Coding Potential Calculator and specific Python scripts in our approach.
Assuntos
RNA Longo não Codificante/genética , Sequência de Bases , Biologia Computacional , DNA Complementar/genética , Zea mays/genéticaRESUMO
Enhanced nitrate removal in the cathode chamber of bioelectrochemical systems (BES) using aerated swine wastewater under high nitrate levels and low organic carbon was investigated in this study, focusing on the relationship between nitrogen and bacterial communities involved in denitrification pathways. BESs with the anion exchange membrane (AEM) under cathodic applied potentials of -0.6 V vs. AgCl/AgCl reference electrode showed a removal rate of 99 ± 2 mg L-1 d-1. Moreover, organic compounds from the untreated full-strength wastewater were simultaneously eliminated in the anode chamber with a removal rate of 0.46 g COD L-1 d-1 with achieved efficiency of 61.4 ± 0.5% from an initial concentration of around 5 g of COD L-1, measured over the course of 7 days. The highest microbial diversity was detected in BESs under potentials of -0.6 V, which include autotrophic denitrifiers such as Syderoxidans, Gallionela and Thiobacillus.
Assuntos
Desnitrificação , Águas Residuárias , Animais , Processos Autotróficos , Reatores Biológicos , Nitratos , Nitrogênio , SuínosRESUMO
BACKGROUND: Cells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control. RESULTS: Here, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1. CONCLUSIONS: We propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas , Células HEK293 , Humanos , Proteína I de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , UbiquitinaçãoRESUMO
Based on recent findings, long noncoding (lnc) RNAs represent a potential class of functional molecules within the cell. In this chapter we describe a computational scheme to identify and classify lncRNAs within maize from full-length cDNA sequences to designate subsets of lncRNAs for which biogenesis and regulatory mechanisms may be verified at the bench. We make use of the Coding Potential Calculator and specific Python scripts in our approach.
Assuntos
DNA Complementar/genética , Genômica/métodos , RNA Longo não Codificante/genética , RNA de Plantas/genética , Zea mays/genética , Genoma de Planta , Humanos , SoftwareRESUMO
BACKGROUND: Computational analysis of cDNA sequences from multiple organisms suggests that a large portion of transcribed DNA does not code for a functional protein. In mammals, noncoding transcription is abundant, and often results in functional RNA molecules that do not appear to encode proteins. Many long noncoding RNAs (lncRNAs) appear to have epigenetic regulatory function in humans, including HOTAIR and XIST. While epigenetic gene regulation is clearly an essential mechanism in plants, relatively little is known about the presence or function of lncRNAs in plants. METHODOLOGY/PRINCIPAL FINDINGS: To explore the connection between lncRNA and epigenetic regulation of gene expression in plants, a computational pipeline using the programming language Python has been developed and applied to maize full length cDNA sequences to identify, classify, and localize potential lncRNAs. The pipeline was used in parallel with an SVM tool for identifying ncRNAs to identify the maximal number of ncRNAs in the dataset. Although the available library of sequences was small and potentially biased toward protein coding transcripts, 15% of the sequences were predicted to be noncoding. Approximately 60% of these sequences appear to act as precursors for small RNA molecules and may function to regulate gene expression via a small RNA dependent mechanism. ncRNAs were predicted to originate from both genic and intergenic loci. Of the lncRNAs that originated from genic loci, â¼20% were antisense to the host gene loci. CONCLUSIONS/SIGNIFICANCE: Consistent with similar studies in other organisms, noncoding transcription appears to be widespread in the maize genome. Computational predictions indicate that maize lncRNAs may function to regulate expression of other genes through multiple RNA mediated mechanisms.