Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress dependent mRNAs

Abstract del trabajo presentado en 12ª Reunión de la Red Española de Levaduras. El Escorial, Madrid.11-13 de diciembre de 2019Pág. 44 del libro de abstracts que se adjunta. RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain

El papel de Mip6 en el metabolismo y la expresión de los tránscritos dependientes de Msn2/4

[ES] La expresión génica en eucariotas es un proceso complejo que comprende diferentes etapas interconectadas, incluyendo la modificación de la cromatina, la transcripción y el procesamiento, exportación al citoplasma, traducción y degradación del ARN. En este estudio, nos centramos en Mip6, que participa en varias etapas del proceso de expresión génica. Mip6 es una proteína que contiene cuatro motivos de reconocimiento de ARN (RRM) y que interacciona con el factor de exportación de ARNm Mex67; en concreto, el triptófano 442 del RRM4 de Mip6 se asocia con el dominio asociado a ubiquitina (UBA) de Mex67. A través de esta interacción, Mip6 tiene un papel en la homeostasis de los tránscritos dependientes de Msn2/4, dos factores de transcripción de respuesta a estrés. En este trabajo, se ha estudiado en profundidad la unión entre Mip6 y Mex67, así como el mecanismo de transporte de Mip6 fuera del núcleo mediado por la secuencia NES y su interacción con los factores de transcripción Msn2/4. La unión entre Mip6 y Mex67 se ve afectada cuando se realizan algunas mutaciones puntuales y deleciones en su secuencia, incluidas mutaciones en la secuencia NES, lo que puede deberse a un reajuste estructural que impediría el acceso al triptófano 442. Sin embargo, la ausencia de Msn5 y Msn2/4 no afecta esta interacción. Por otro lado, se ha optimizado el protocolo de la tecnología CRISPR en levadura con la creación del mutante ADH1pr-Mip6W442A-GFP, que se ha visto que también afecta a la interacción de Mip6 y Mex67. Además, con el estudio de esta nueva cepa, se ha observado una diferencia en el fenotipo de crecimiento cuando Mip6 está sobreexpresada, lo que puede indicar que los niveles de la proteína son importantes en su función dentro de la célula.[EN] Eukaryotic gene expression is a complex process which comprises different interconnected steps, including chromatin modification, transcription and RNA processing, export to cytoplasm, translation and degradation. In this study, we focus on Mip6, which participates in different steps of the gene expression process. Mip6 is a protein which contains four RNA recognition motifs (RRM) and interacts with the mRNA export factor Mex67; specifically, tryptophan 442 (W442) of Mip6-RRM4 associates with the ubiquitin-associated (UBA) domain of Mex67. Through this interaction, Mip6 has a role in the homeostasis of the transcripts that depend on Msn2/4, two stress-response transcription factors. In this work, we study the Mip6-Mex67 binding in depth, as well as the Mip6 NES-mediated export mechanism, and its interaction with the transcription factors Msn2/4. The Mip6-Mex67 binding is impaired when some point mutations and deletions are performed in the sequence, including the putative NES sequence, what may be due to a structural rearrangement that affects W442 accessibility. However, the lack of Msn5 and Msn2/4 does not affect this interaction. On the other hand, CRISPR technology in yeast protocol has been tuned by creating an ADH1pr-Mip6W442A-GFP mutant, which presents the phenotype of impairing the Mip6-Mex67 interaction. Furthermore, the study of this new mutant strain has reported a different growth phenotype when Mip6 is overexpressed, suggesting the importance of the protein levels in its role within the cell.Tejada Colón, A. (2019). The Role of MIP6 in the Metabolism and Expression of MSN2/4-Dependent Transcripts. http://hdl.handle.net/10251/124562TFG

Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress-dependent mRNAs

21 páginas, 8 figuras. PAR-CLIP datasets are available at GEO as GSE126236: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126236RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.MM-E, CN-C and AT-C were supported by the GVA (Val I+D: ACIF/2015/025) PROMETEO (PROM/2016/093) and GVA (Val I+D: ACIF/2019/212). NM was partially supported by the European Union Erasmus Mundus programme. NMR experiments were performed in the "Manuel Rico" NMR laboratory (LMR) (CSIC). The authors would like to thank Diamond Light Source for beamtime (proposal mx-10121), and the staff of beamline I04 for assistance with crystal testing and data collection. This study was supported by funds to SR-N from the Spanish MINECO, MICIIN (BFU2014-57636, BFU2015-71978, PGC2018- 099872-B-I00) and the Generalitat Valenciana (PROM/2012/061, ACOMP2014/061 and PROMETEO 2016/093), and to JB from the Spanish MINECO (SAF2015-67077-R, SAF2017-89901-R) and JMP-C from the Spanish MINECO (CTQ2018-84371) and Comunidad de Madrid (B2017/BMD-3770).Peer reviewe