Identification of karyopherins involved in the nuclear import of RNA exosome subunit Rrp6 in Saccharomyces cerevisiae

Abstract

The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In , Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.

Distinct and evolutionary conserved structural features of the human nuclear exosome complex

“…In yeast, there is a single yRrp44 protein that is part of both the nuclear and cytoplasmic forms of the exosome. The current view is that yeast yRrp44 assembles together with yExo-9 in the cytoplasm and is transported to the nucleus by the import capacity of yRrp6 ( Gonzales-Zubiate et al, 2017 ). Instead, the situation is more complex in human cells, as there are instead two different paralogues (hDIS3 and hDIS3L) that have to be selectively incorporated in the nuclear and cytoplasmic forms of the complex ( Tomecki et al, 2010 ).…”

“…The nuclear exosome includes four specific cofactors that are conserved from lower to higher eukaryotes. In yeast, the yRrp6 exoribonuclease and its binding partner yRrp47 are tethered with high affinity to the cap of yExo-9 ( Kowalinski et al, 2016 ; Makino et al, 2015 ; Zinder et al, 2016 ) and target the complex to the nuclear compartment ( Gonzales-Zubiate et al, 2017 ). The yRrp6-yRrp47 dimer together with another stably associated subunit, yMpp6, recruits the essential 3'−5' RNA helicase yMtr4 ( Falk et al, 2017 ; Schuch et al, 2014 ; Wasmuth et al, 2017 ).…”

Regulation of the conserved 3′‐5′ exoribonuclease EXOSC10/Rrp6 during cell division, development and cancer

“…Third, Rrp6 is a substrate for protein SUMOylation and Rrp6 interacts directly with suppressor of mif two 3 (Smt3), a protein of the SUMO family (Wohlschlegel et al ., 2004; Gonzales‐Zubiate et al ., 2017; Pabst et al ., 2019). During this modification process SUMO proteins are attached to their targets by multi‐component complexes.…”

“…Ubiquitination requires so‐called E3 ligases (writers), ubiquitin‐binding effectors (readers), and deubiquitylases (erasers) that work together to establish the protein concentration, conformation and localization needed for robust cell growth and development (Oh, Akopian & Rape, 2018). Rrp6 physically interacts with binds ubiquitin ligase 2 (Bul2), a subunit of the reverses spt phenotype 5 (Rsp5) E3‐ubiquitin ligase important for growth during stress (Kaida, Toh‐e & Kikuchi, 2003; Frattini et al ., 2017), the ubiquitin‐activating enzyme (E1) Uba1, the ubiquitin conjugating enzyme (E2) Ubc1, which is associated with the APC/C (Girard, Tenthorey & Morgan, 2015; Gonzales‐Zubiate et al ., 2017) and Grr1, a glucose‐responsive subunit of the Skp1‐Cullin‐F‐box protein (SCF) ubiquitin‐ligase complex (Fey & Lanker, 2007; Gonzales‐Zubiate et al ., 2017).…”

Caracterização da função molecular de Nop53 e de seu papel no controle do exossomo em <i>Saccharomyces cerevisiae</i>

“…(b) Comparação de contagem de espectros identificados por Espectrometria de Massas do exossomo na presença (galactose) e ausência (glicose) de Nop53. et al, 2013; Gonzales-Zubiate et al, 2017). Como mostrado aqui, Rrp43 co-purifica mais proteínas citoplasmáticas envolvidas em outras etapas de processamento e/ou degradação de RNAs (Tabela 4.1), interações que não são afetadas por Nop53, uma proteína nucleolar.…”