The multi PAM2 protein Upa2 functions as novel core component of endosomal mRNA transport

Abstract

<jats:title>Abstract</jats:title><jats:p>mRNA transport determines spatiotemporal protein expression. Transport units are higher-order ribonucleoprotein complexes containing cargo mRNAs, RNA-binding proteins and accessory proteins. Endosomal mRNA transport in fungal hyphae belongs to the best-studied translocation mechanisms. Although several factors are known, additional core components are missing. Here, we describe the 232 kDa protein Upa2 containing multiple PAM2 motifs (poly[A]-binding protein [Pab1] associated motif 2) as a novel core component. Loss of Upa2 disturbs transport of cargo mRNAs and associated Pab1. Upa2 is present on almost all transport endosomes in an mRNA dependent-manner. Surprisingly, all four PAM2 motifs are dispensable for function during unipolar hyphal growth. Instead, Upa2 harbours a novel N-terminal effector domain as important functional determinant as well as a C-terminal GWW motif for specific endosomal localisation. In essence, Upa2 meets all the criteria of a novel core component of endosomal mRNA transport and appears to carry out crucial scaffolding functions.</jats:p>

Ustilago maydis Serves as a Novel Production Host for the Synthesis of Plant and Fungal Sesquiterpenoids

“…Microscopy was carried out as described before (Baumann et al, 2016;Jankowski et al, 2019) using two systems: (i) a wide-field microscope set-up from Visitron Systems (Puchheim, Germany), Axio Imager M1 equipped with a Spot Pursuit CCD camera (Diagnostic Instruments, Sterling Heights, MI, the USA) and the objective lens Plan Neofluar (40×, NA 1.3; 63×, NA 1.25; Carl Zeiss, Jena, Germany). The excitation of fluorescently labeled proteins was carried out using an HXP metal halide lamp (LEj, Jena, Germany) in combination with a filter set for green fluorescent protein (ET470/40BP, ET495LP and ET525/50BP) and Hoechst 33342 dye (HC387/11BP, BS409LP and HC 447/60BP; AHF Analysentechnik, Tübingen, Germany).…”

Ustilago maydisserves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

“…Microscopy was carried out as described before (Baumann et al, 2016;Jankowski et al, 2019) using two systems: (i) a wide-field microscope set-up from Visitron Systems (Puchheim, Germany), Axio Imager M1 equipped with a Spot Pursuit CCD camera (Diagnostic Instruments, Sterling Heights, MI, USA) and the objective lens Plan Neofluar (40 ×, NA 1.3; 63 ×, NA 1.25; Carl Zeiss, Jena, Germany). The excitation of fluorescently labelled proteins was carried out using an HXP metal halide lamp (LEj, Jena, Germany) in combination with a filter set for green fluorescent protein (ET470/40BP, ET495LP and ET525/50BP) and Hoechst 33342 dye (HC387/11BP, BS409LP and HC 447/ 60BP; AHF Analysentechnik, Tübingen, Germany).…”

“…(ii) In order to record fluorescence signals localised in subcellular compartments with higher sensitivity, laser-based epifluorescence microscopy was performed on a Zeiss Axio Observer.Z1 equipped with CoolSNAP HQ2 CCD (Photometrics, Tuscon, AZ, USA) and ORCA-Flash4.0 V2 + CMOS (Hamamatsu Photonics Deutschland GmbH, Geldern, Germany) cameras. The microscopy set-up was the same as described above (Jankowski et al, 2019). For excitation, a VS-LMS4 Laser-Merge-System (Visitron Systems) that combines solid-state lasers for excitation of Gfp (488 nm at 50 or 100 mW) was used.…”

Inside-Out: From Endosomes to Extracellular Vesicles in Fungal RNA Transport

“…Upa2, which exceptionally contains four PAM2 motifs, shuttles on almost all Rrm4-positive endosomes and is important for efficient unipolar hyphal growth (Jankowski et al, 2019).…”

“…Thus, Upa2 classifies as a novel core component of endosomal mRNA transport, which most likely serves as a scaffold protein for endosomal mRNP assembly or stability during transport ( Fig. 1E; Jankowski et al, 2019).…”