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.

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.

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.

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].

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.

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.