Proposal for a scientific collaboration between the labs of Rodríguez-Navarro (SRN) , Bravo (JB) y Pérez-Cañadillas (JMPC).

cerevisiae Mip6 (Mex67 interacting protein 6) is a multidomain protein predicted to have up to four RRM (RNA binding domains) probably organized in two tandems. Susana Rodriguez Navarro´s lab has been studying this protein and its connection with the formation of stress granules during the last years. As part of this research they have established collaboration with the group of Jerónimo Bravo for the resolution of the 3D structures of Mip6 and associated complexes by X-ray crystallography and currently they have produced the X-ray structures of the third RRM domain of Mip6 and the C-terminal domain of Mex67 that interacts with Mip6. Few years ago the JMPC group made some exploratory experiments with constructs of Mip6 and its paralog Pes4, aiming for structural studies by NMR. However all the constructs prove to be very insoluble. However the SRN and JB groups have been able to overcome these problems and are capable to produce soluble constructs of RRM3, RRM4 and RRM3-4. Nevertheless they still have not been able to obtain structures of RRM4, RRM3-4 or the complexes with Mex67 C-terminal domain.

The collaboration with the JMPC group seeks to expand the field of study of this project by incorporating an alternative method (NMR) to map the interaction between Mip6 and Mex67. The objective is to obtain the assignments of Mip6 RRM4 and the chemical shift map of the interaction of this module with Mex67. Once obtained the data would be use to generate structural docking models using the crystallography data from JB group and the interaction model will be validated by biochemical or cell biology data by the SRN group, which acts as coordinator of the project.

Follow up meeting between the labs of Rodríguez-Navarro (SRN), Bravo (JB) y Pérez-Cañadillas (JMPC) to discuss recent results on the Mip6/Mex67 system.

Last April we had a meeting to establish a scientific collaboration between our labs. That meeting was supported by the BFU2015-71978-REDT project. We planned a series of specific experiments to further prove the interaction between yeast proteins Mip6 and Mex67. The objectives of the proposal (see previous document) have been fulfilled and now we have new NMR data that map the interaction interface and that allow a rational design of mutants to test mechanistic hypothesis in vivo. We need to held a new meeting between our three labs to discuss the exploitation strategy of these results and for further planning.

The collaboration with the JMPC group seeks to expand the field of study of this project by incorporating an alternative method (NMR) to map the interaction between Mip6 and Mex67. The objective is to obtain the assignments of Mip6 RRM4 and the chemical shift map of the interaction of this module with Mex67. Once obtained the data would be use to generate structural docking models using the crystallography data from JB group and the interaction model will be validated by biochemical or cell biology data by the SRN group, which acts as coordinator of the project.

Follow up meeting between the labs of Rodríguez-Navarro (SRN), Bravo (JB) y Pérez-Cañadillas (JMPC) to discuss recent results on the Mip6/Mex67 system.

Last April we had a meeting to establish a scientific collaboration between our labs. That meeting was supported by the BFU2015-71978-REDT project. We planned a series of specific experiments to further prove the interaction between yeast proteins Mip6 and Mex67. The objectives of the proposal (see previous document) have been fulfilled and now we have new NMR data that map the interaction interface and that allow a rational design of mutants to test mechanistic hypothesis in vivo. We need to held a new meeting between our three labs to discuss the exploitation strategy of these results and for further planning.

The collaboration with the JMPC group seeks to expand the field of study of this project by incorporating an alternative method (NMR) to map the interaction between Mip6 and Mex67. The objective is to obtain the assignments of Mip6 RRM4 and the chemical shift map of the interaction of this module with Mex67. Once obtained the data would be use to generate structural docking models using the crystallography data from JB group and the interaction model will be validated by biochemical or cell biology data by the SRN group, which acts as coordinator of the project.

A proposal of scientific collaboration between labs of J Pérez and C Suñé NMR studies of the WW and FF domains of TCERG1 and PRPF40B

A proposal of scientific collaboration between the labs of Olga Calvo and José E. Pérez Ortín

is an evolutionary conserved factor that was originally described as a transcriptional stimulatory protein based on its capacity to bind the general transcription factor TFIIB and its homology to human positive coactivator PC4 [1]. Recently, orthologues of PC4/Sub1 have been also identified in prokaryotes [2].

has been implicated in many steps of mRNA metabolism: (I) It is a functional component of the preinitiation complex [3]. (II) ySub1/hPC4 links transcription initiation to termination and 3′-end processing, via the interaction with a polyadenylation factor, and this interaction is evolutionary conserved in human cells [4]. (III) Sub1 associates with Spt5 to influence transcription elongation rate [5] and it globally modulates RNAP II-CTD phosphorylation [6]. (IV) Lately, it has been proposed a role for Sub1 during osmotic and oxidative stress [7,8].  Moreover, both Sub1 and PC4, also play a role in transcription by RNAPIII [9,10]. The laboratory of O. Calvo has significantly contributed to the characterization of many of the Sub1 functions. However, the effects of Sub1 in the genome-wide expression is unknown 

A proposal of scientific collaboration between the labs of Sebastián Chávez and Carles Suñé

Prefoldin was described as a heterohexameric cytoplasmic complex, conserved from archaea to higher eukaryotes, and involved in actin and tubulin biogenesis (Reviewed in 1). Chávez’s lab has reported that, in addition to its cytoplasmic role, yeast prefoldin is also localized in the nucleus, and recruited to coding regions in a transcription-dependent manner (2). We also reported that the pfd1Δ mutant, lacking one of the subunits of the complex, exhibits transcriptional defects and impaired histone eviction during transcription elongation. We also obtained evidence supporting a direct role of prefoldin on the transcription of chromatin templates in vitro.

In order to increase our knowledge of the precise role of prefoldin in gene expression we have performed additional in vitro and in vivo studies in human cells.

We have found that depletion of human prefoldin also alters RNA pol II-dependent transcription. For instance, under prefoldin-depleted conditions Ser2-phosphorylation of RNA pol II CTD drops across the transcribed region of all genes that we have tested so far. Similar results were obtained in a CRISPRed cell line, depleted for PFDN5 (one of the human prefoldin subunits).

We wondered if cotranscriptional pre-mRNA splicing was also affected and found a clear delay in coupling between transcription of DRB-synchronized RNA pol II populations and intron removal from pre-mRNA. We have also found general alterations in the patterns of mRNA isoforms in total RNA-seq analyses.

In order to confirm and characterize this pre-mRNA splicing defects, we have designed experiments to be performed in Carles Suñé lab (Instituto de Parasitología y Biomedicina López Neyra). The alternative splicing of several reporter minigene constructs (Bcl-x and Fas/CD95) will be tested upon prefoldin depletion using engineered CRISPR/cas9 cells. Briefly, cells will be transfected with the reporter minigenes and harvested 24 or 48 h later. Total RNA will be extracted and semi- and quantitative PCR will be carried out to amplify the specific transcripts. At least, three independent experiments will be performed.

This task is included within the following scientific goals of the mRNALife “red temática”:

  • Advances in the knowledge of the biology of the mechanisms that regulate the interconnections between RNA processes

A proposal of scientific collaboration between the labs of Olga Calvo and José E. Pérez Ortín

The laboratories of Olga Calvo and José E. Pérez Ortín started collaboration on the transcriptomic analysis of several transcription factors in order to cast light on their genome-wide effects. We are now specifically focused on studying the role of Rpb4 in gene looping and in the transcriptional directionality. We are very interested on using the GRO-seq technique, developed in J.E. Pérez Ortín Lab, to try to understand the effects of rpb4 deletion on those processes. For that purpose, we request the following financial support:

Costs of two days visit of Olga Calvo to the lab of José E. Pérez Ortín in order to plan the specific details of the Project.

This task could be included within the scientific goal of the “Red Temática”:

  •  Cuestiones tecnológicas: Avances en la utilización de recursos tecnológicos.
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