Study of the cellular transport mechanism of Exosome complex subunits in Saccharomyces cerevisiae

Author(s)

Ellen Kazumi Okuda

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2020-11-17

Examining board members

Carla Columbano de Oliveira; Nathalie Cella; Nicolas Carlos Hoch; Nilson Ivo Tonin Zanchin

• Advisor: Carla Columbano de Oliveira

Abstract

Exosome is a multiprotein complex responsible for processing and degradation of many classes of RNAs in eukaryotic cells. This complex localizes both to the nucleus and cytoplasm. Exosome complex has two catalytic subunits: Rrp6, which is exclusively nuclear; and Rrp44, that has endonucleolitic and exonucleolitic domains, being essential for cell growth. The aim of this project was to analyze the localization of the exosome and the nuclear transport pathways of its subunits, focusing on Rrp44. For that, the main technique used was fluorescent microscopy, which allowed the analysis in several different strains and protein fusions with GFP and mCherry. The results show a subnucleolar localization of the exosome complex when its subunits are expressed at endogenous levels, under control of their own promoters, fused to GFP. Interestingly, the subunits Rrp6 and Rrp44 still localize to the nucleus when overexpressed, whereas other exosome subunits, such as Rrp41 and Rrp43 show strong signals in the cytoplasm when they are overexpressed in the same conditions. Moreover, we could identify the putative nuclear localization signals of Rrp44, which are recognized by the essential karyopherins Srp1 and Kap95, the main karyopherins affecting the nuclear transport of Rrp44. The results of this work show that the yeast exosome is mainly localized to the nucleus and concentrated in the nucleolus, which is particularly important for the functional study of this complex. (AU)

• FAPESP's process: 17/17777-9 - Study of the celular transport mechanism of the exosome's subunits in Saccharomyces cerevisiae
• Grantee: Ellen Kazumi Okuda
• Support Opportunities: Scholarships in Brazil - Master