95 research outputs found
The translational landscape of the splicing factor SRSF1 and its role in mitosis
The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is
a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene
network that is translationally regulated by SRSF1, we performed a high-throughput deep
sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified
approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding
proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which
are essential for accurate chromosome segregation. Indeed, we show that translational activity of
SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display
alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly
suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on
the complex role of SRSF1 in the control of gene expression at multiple levels and its implications
in cancer
Regulation and function of AGR2 and p53 pathways
Inactivation of p53 by mutation occurs in half of human tumours. The
majority of these mutations affect the DNA-binding core domain and hence impair
DNA binding and hinder transcription of p53 target genes. A wealth of data indicates
that even cancers carrying wild type p53 protein, evolve mechanisms to render the
p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or
the alternative mechanisms, allows unpurturbed tumour growth. Recent work
identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53
expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway.
In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion
potentiates p53-dependent DNA damage response. As there were no physiological
signals known that regulate the AGR2-p53 axis, here I set out to identify pathways
that activate or inhibit AGR2. I found that transforming growth factor β(TGF-
β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and
increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4-
dependent manner and triggers AGR2 protein degradation involving an ATM kinase.
I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent
AGR2 degradation. Interestingly, SNIP1 overexpression by itself
promoted AGR2 protein degradation. I found that AGR2 protein degradation was
proteasome independent and involed autophagy-lysosomal degradation pathway. As
the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying
interactors of AGR2 may potentially further our understanding of the mechanism
accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein
Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2
binding partner. Mutations of the two ATP binding motifs in Reptin resulted in
altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin
complex stability. I also identified the Reptin docking site and it was mapped to a
divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated
with the p53 protein. Subsequently, I showed that Reptin protein
can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I
found that Reptin formed a stable complex, independent of AGR2, with p53 R175H,
p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin
function in cancers bearing mutant form of p53 protein
Die Rolle des Protein C-Mangels, des Protein S-Mangels und des Antithrombin-Mangels bei dem Organverlust nach einer allogenen Nierentransplantation
Im Rahmen dieser Studie wurden 161 Patienten mit terminaler Niereninsuffizienz direkt vor und 365 Tage nach Nierentransplantation auf Parameter der Gerinnung und Fibrinolyse untersucht. Von diesen Patienten kam es bei 16 während der Beobachtungszeit zu einer Abstoßung mit konsequtivem Organverlust. Bei keinem der Patienten lag ein angeborener Mangel an Protein C, Protein S oder Antithrombin vor; bei 5 wurde ein erworbener Protein C-Mangel diagnostiziert, bei keinem der Patienten kam es zu einem Organverlust; bei 7 wurde ein Protein S-Mangel festgestellt, davon verloren 2 ihre Transplantate; bei 4 wurde ein Antithrombin-Mangel diagnostiziert, aus dieser Gruppe erlitt ein Patient einen Organverlust. Im Rahmen dieser Studie konnte gezeigt werden, dass diese durch Nierenerkrankungen erworbene Mangelzustände sich als nicht signifikante prothrombotische Risikofaktoren für Transplantatverluste erwiesen
Two Years of Experience in Implementation of the mobiREH Remote Rehabilitation System Supporting Patients and Physiotherapists
The aim of this work is to characterise the process of developing the mobiREH telemedical rehabilitation system which was created as a result of cooperation between the mReh start-up team and the scientific team from the Academy of Physical Education in Kraków. The most significant global and local challenges for rehabilitative health services are: the increasing number of patients awaiting rehabilitation, the increasing waiting time for rehabilitation, and the decreasing number of medical specialists. A mobiREH rehabilitation system is a system that supports home based rehabilitation and helps resolve these problems. The system consists of (a) a mobile application, (b) wearable sensors for patients, and (c) a web-based platform for medical specialists
Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function
Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis
Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner
Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.Work at the M.G. lab was supported by the Spanish Ministry of Sciences and Innovation (BFU2016-78849-P and PID2019-105949GB-I00, co-financed by the European Union FEDER funds), a CSIC grant (2019AEP004), and a Salvador de Madariaga mobility grant (PRX19/00293). J.M.F.-J., C.S.-M., and J.I.-A. were supported by the Spanish Ministry of Sciences and Innovation fellowships (BES-2014-070050, BES-2017-079897, and PRE2020-095071, respectively); S.M.-V. was supported by a predoctoral fellowship from the Spanish Ministry of Education and Universities (FPU18/04794); and M.S.-P. was supported by an AGAUR-FI predoctoral fellowship co-financed by Generalitat de Catalunya and the European Social Fund. A.J. was supported by the Spanish Ministry of Sciences and Innovation (BFU2017-82805-C2-1-P and PID2020-112783GB-C21) and J.F.C. by core funding to the MRC Human Genetics Unit from the Medical Research Council (UK)
Splicing factors Sf3A2 and Prp31 have direct roles in mitotic chromosome segregation
Several studies have shown that RNAi-mediated depletion of splicing factors (SFs) results in mitotic abnormalities. However, it is currently unclear whether these abnormalities reflect defective splicing of specific pre-mRNAs or a direct role of the SFs in mitosis. Here, we show that two highly conserved SFs, Sf3A2 and Prp31, are required for chromosome segregation in both Drosophila and human cells. Injections of anti-Sf3A2 and anti-Prp31 antibodies into Drosophila embryos disrupt mitotic division within 1 min, arguing strongly against a splicing-related mitotic function of these factors. We demonstrate that both SFs bind spindle microtubules (MTs) and the Ndc80 complex, which in Sf3A2- and Prp31-depleted cells is not tightly associated with the kinetochores; in HeLa cells the Ndc80/HEC1-SF interaction is restricted to the M phase. These results indicate that Sf3A2 and Prp31 directly regulate interactions among kinetochores, spindle microtubules and the Ndc80 complex in both Drosophila and human cells
A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes
The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII. We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.Fil: Maslon, M.. University of Edinburgh; Reino UnidoFil: Braunschweig, U.. University of Toronto; CanadáFil: Aitken, S.. University of Edinburgh; Reino UnidoFil: Mann, A.R.. University of Edinburgh; Reino UnidoFil: Kilanowski, F.. University of Edinburgh; Reino UnidoFil: Hunter, C.H.. University of Edinburgh; Reino UnidoFil: Blencowe, B.J.. University of Toronto; CanadáFil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones CientÃficas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de FisiologÃa, BiologÃa Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de FisiologÃa, BiologÃa Molecular y Neurociencias; ArgentinaFil: Adams, I.. University of Edinburgh; Reino UnidoFil: Cáceres, J.F.. University of Edinburgh; Reino Unid
Gene Profiling of Mta1 Identifies Novel Gene Targets and Functions
BACKGROUND: Metastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1. METHODS: Gene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes. SIGNIFICANCE/CONCLUSION: This study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define "bona fide" Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions
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