RESUMEN
Spinal muscular atrophy (SMA) is an inherited neurodegenerative condition caused by a reduction in the amount of functional survival motor neuron (SMN) protein. SMN has been implicated in transport of mRNA in neural cells for local translation. We previously identified microtubule-dependent mobile vesicles rich in SMN and SNRPB, a member of the Sm family of small nuclear ribonucleoprotein (snRNP)-associated proteins, in neural cells. By comparing the interactomes of SNRPB and SNRPN, a neural-specific Sm protein, we now show that the essential neural protein neurochondrin (NCDN) interacts with Sm proteins and SMN in the context of mobile vesicles in neurites. NCDN has roles in protein localisation in neural cells and in maintenance of cell polarity. NCDN is required for the correct localisation of SMN, suggesting they may both be required for formation and transport of trafficking vesicles. NCDN may have potential as a therapeutic target for SMA together with, or in place of the targeting of SMN expression.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Atrofia Muscular Espinal/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Complejo SMN/metabolismo , Células Cultivadas , HumanosRESUMEN
From their initial identification as 'nucleolar accessory bodies' more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.
Asunto(s)
Nucléolo Celular/metabolismo , Cuerpos Enrollados/metabolismo , Animales , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Homeostasis del TelómeroRESUMEN
The biogenesis of splicing snRNPs (small nuclear ribonucleoproteins) is a complex process, beginning and ending in the nucleus of the cell but including key stages that take place in the cytoplasm. In particular, the SMN (survival motor neuron) protein complex is required for addition of the core Sm proteins to the snRNP. Insufficiency of SMN results in the inherited neurodegenerative condition, spinal muscular atrophy (SMA). Details of the physical organization of the cytoplasmic stages of snRNP biogenesis are unknown. Here, we use time-resolved quantitative proteomics to identify proteins that associate preferentially with either newly assembled or mature splicing snRNPs. We identified highly mobile SmB protein-trafficking vesicles in neural cells, which are dependent on the cellular levels of SMN and SmB for their morphology and mobility. We propose that these represent a family of related vesicles, some of which play a role in snRNP biogenesis and some that might play more diverse roles in cellular RNA metabolism.
Asunto(s)
Neuritas/metabolismo , Proteoma/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteínas Nucleares snRNP/metabolismo , Dineínas/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Neuronas/metabolismo , Transporte de Proteínas , Proteómica , Empalme del ARN , Imagen de Lapso de Tiempo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
DM1 (myotonic dystrophy type 1) is caused by elongation of a CTG repeat in the DMPK (dystrophia myotonica-protein kinase) gene. mRNA transcripts containing these CUGexp (CUG expansion) repeats form accumulations, or foci, in the nucleus of the cell. The pathogenesis of DM1 is proposed to result from inappropriate patterns of alternative splicing caused by sequestration of the developmentally regulated alternative splicing factor MBNL1 (muscleblind-like 1) by these foci. Since eye lens cataract is a common feature of DM1 we have examined the distribution and dynamics of MBNL1 in lens epithelial cell lines derived from patients with DM1. The results of the present study demonstrate that only a small proportion of nuclear MBNL1 accumulates in CUGexp pre-mRNA foci. MBNL1 is, however, highly mobile and changes localization in response to altered transcription and splicing activity. Moreover, immunolocalization studies in lens sections suggest that a change in MBNL1 distribution is important during lens growth and differentiation. Although these data suggest that the loss of MBNL1 function due to accumulation in foci is an unlikely explanation for DM1 symptoms in the lens, they do demonstrate a strong relationship between the subcellular MBNL1 localization and pathways of cellular differentiation, providing an insight into the sensitivity of the lens to changes in MBNL1 distribution.
Asunto(s)
Cristalino/citología , Cristalino/crecimiento & desarrollo , Distrofia Miotónica/genética , Distrofia Miotónica/patología , Proteínas de Unión al ARN/genética , Transcripción Genética/genética , Empalme Alternativo/genética , Animales , Células Cultivadas , Células Epiteliales/fisiología , Humanos , Simulación de Dinámica Molecular , Fracciones Subcelulares/fisiología , PorcinosRESUMEN
RNA regulation in mammalian cells requires complex physical compartmentalisation, using structures thought to be formed by liquid-liquid phase separation. Disruption of these structures is implicated in numerous degenerative diseases. Myotonic dystrophy type 1 (DM1) is a multi-systemic trinucleotide repeat disorder resulting from an expansion of nucleotides CTG (CTGexp) in the DNA encoding DM1 protein kinase (DMPK). The cellular hallmark of DM1 is the formation of nuclear foci that contain expanded DMPK RNA (CUGexp) (with thymine instead of uracil). We report here the deregulation of stress granules (SGs) and processing bodies (P-bodies), two cytoplasmic structures key for mRNA regulation, in cell culture models of DM1. Alterations to the rates of formation and dispersal of SGs suggest an altered ability of cells to respond to stress associated with DM1, while changes to the structure and dynamics of SGs and P-bodies suggest that a widespread alteration to the biophysical properties of cellular structures is a consequence of the presence of CUGexp RNA.
Asunto(s)
Distrofia Miotónica , Animales , Mamíferos/metabolismo , Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismo , Cuerpos de Procesamiento , ARN , Gránulos de Estrés , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady-state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo. The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH-SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue-specific pathology of SMA.
Asunto(s)
Diferenciación Celular , Núcleo Celular/metabolismo , Neuroblastoma/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Línea Celular Tumoral , Cuerpos Enrollados/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunohistoquímica , Microinyecciones , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Neuroblastoma/genética , Plásmidos , Transporte de Proteínas/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Factores de Tiempo , Transfección , Proteínas Nucleares snRNP/metabolismoRESUMEN
Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.
Asunto(s)
Microsomas Hepáticos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Nucleótidos/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Animales , Isótopos de Carbono , Clonación Molecular , Cricetinae , Cricetulus , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Glucuronosiltransferasa/metabolismo , Células HeLa , Humanos , Cinética , Neoplasias Hepáticas Experimentales , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Nucleótidos/genética , Ratas , Uridina Difosfato Ácido Glucurónico/farmacocinética , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Eukaryotic cells enclose their genome within a dedicated organelle, the nucleus, which is the site of major cellular events such as messenger RNA synthesis and processing, ribosome subunit biogenesis and DNA replication. Like the cytoplasm, the nucleus is compartmentalized to facilitate efficient coordination of these pathways, although subnuclear compartments form without the use of membranes. Numerous disease states have been linked to dysfunction of these compartments or 'nuclear bodies'. Recent advances have shed light on the formation and maintenance of key structures, including nucleoli, splicing speckles, paraspeckles, Cajal bodies, histone locus bodies and promyelocytic leukemia bodies. Here, we review the impact of these findings, which provide major insights into dynamic processes that affect both structure and function within the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Animales , Núcleo Celular/genética , Epigénesis Genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN/metabolismo , Telomerasa/metabolismo , Telómero/metabolismoRESUMEN
We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.
Asunto(s)
Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , Cartilla de ADN , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HeLa , Humanos , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Empalme del ARN , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Empalme U2AF , TransfecciónRESUMEN
The cell is the basic building block of human life. Each of us has existed as a single cell--the fertilized egg--and each of us is made up of billions of cells specialized in many different ways to form our tissues and organs. The nucleus of the cell, described as far back as 1682, is known to be the site of storage of chromosomes that carry the essential and unique DNA blueprint for life. With the recent publication of the entire human genome, our knowledge of exactly what our genes say has increased immeasurably. This, however, is only a small part of the story. In order for the chromosomal genes to function correctly, a complex cellular machinery must rewrite (or transcribe) the genetic instructions of the DNA into a temporary messenger molecule, messenger RNA (mRNA), rearrange (or splice) this message into a readable format and then produce a protein that accurately represents the DNA code. It is these protein molecules that are the functional result of the genetic information. This whole process is termed 'gene expression'. Both transcription and splicing of the mRNA message are carried out in the nucleus. These events must be performed accurately and efficiently in a minute volume already full of highly packaged DNA. An ever-increasing number of sub-nuclear structures have been described, from the nucleolus (first described in 1835) to newly discovered 'paraspeckles' and 'clastosomes'. In fact, as increasing numbers of molecular probes become available, so the complexity of nuclear structure appears to expand. The functions of some of these structures are currently unknown. Those whose functions are, at least partly, understood play roles in gene expression. Interestingly, alterations in nuclear structure are associated with human diseases such as spinal muscular atrophy and promyelocytic leukaemia, suggesting that the control of nuclear organization may be vital to health. The dynamic nature of the structure of the mammalian nucleus has come under increasing scrutiny over the past few years. This has largely been driven by advances in microscopy as well as the advent of in vivo labelling techniques for sub-nuclear structures. It is now possible, using a protein originally isolated from jellyfish, to visualize sub-nuclear structures in living cultured cells. Together with three-dimensional time-lapse microscopy and an ever-expanding range of photo-bleaching techniques, this technology allows us to ask detailed questions about movements of sub-nuclear structures themselves and of the proteins contained within them. It has recently become clear that sub-nuclear structures are capable of moving within the nucleus and of physically interacting with each other. It is also now known that there is a constant flux of molecules into and out of these mobile structures as well as exchange of molecules between them, rather like passengers travelling on the London Underground. The challenge for the future is to relate dynamic events at the microscopic and molecular levels back to the organism as a whole. Only by understanding how the information encoded on genes is accurately expressed at the right time and in the right place can we really take advantage of the knowledge currently available to us.
Asunto(s)
Núcleo Celular/genética , Núcleo Celular/ultraestructura , ADN/genética , ADN/ultraestructura , Movimiento/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Animales , Humanos , Mamíferos , Proteínas Motoras Moleculares/fisiología , Proteínas Motoras Moleculares/ultraestructura , Proteínas Nucleares/metabolismoRESUMEN
Analysis of stable cell lines expressing fluorescently tagged survival of motor neurons protein (SMN) and coilin shows striking differences in their dynamic behaviour, both in the nucleus and during mitosis. Cajal bodies labelled with either FP-SMN or FP-coilin show similar behaviour and frequency of movements. However, fluorescence recovery after photobleaching (FRAP) studies show that SMN returns approximately 50-fold more slowly to Cajal bodies than does coilin. Time-lapse studies on cells progressing from prophase through to G1 show further differences between SMN and coilin, both in their localisation in telophase and in the timing of their re-entry into daughter nuclei. The data reveal similarities between Cajal bodies and nucleoli in their behaviour during mitosis. This in vivo study indicates that SMN and coilin interact differentially with Cajal bodies and reveals parallels in the pathway for reassembly of nucleoli and Cajal bodies following mitosis.
Asunto(s)
Cuerpos Enrollados/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Autoantígenos , Proteínas Bacterianas/metabolismo , Línea Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Separación Celular , Citometría de Flujo , Fase G1 , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Immunoblotting , Interfase , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mitosis , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Proteínas Nucleares snRNPRESUMEN
Protein phosphatase 4 (PPP4) is a ubiquitous essential protein serine/threonine phosphatase found in higher eukaryotes. Coordinate variation of the levels of the catalytic subunit (PPP4c) and the regulatory subunit (R2) suggests that PPP4c and R2 form a heterodimeric core to which other regulatory subunits bind. Two proteins that specifically co-purify with Flag-epitope-tagged R2 expressed in HEK-293 cells were identified as Gemin3 and Gemin4. These two proteins have been identified previously as components of the Survival of Motor Neurons (SMN) protein complex, which is functionally defective in the hereditary disorder spinal muscular atrophy. Immuno-sedimentation of the epitope-tagged SMN protein complex from HeLa cells expressing CFP-SMN showed that the SMN protein interacts, as previously reported, with Gemin2 (SIP1), Gemin3 and Gemin4 and in addition associates with PPP4c. The SMN complex has been implicated in the assembly and maturation of small nuclear ribonucleoproteins (snRNPs). Expression of GFP-R2-PPP4c in HeLa cells enhances the temporal localisation of newly formed snRNPs, which is consistent with an association of R2-PPP4c with the SMN protein complex.