N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements

Abstract

RNA binding proteins associated with amyotrophic lateral sclerosis (ALS) and muscle myopathy possess sequence elements that are low in complexity, or bear resemblance to yeast prion domains. These sequence elements appear to mediate phase separation into liquid-like membraneless organelles. Using fusion proteins of matrin 3 (MATR3) to yellow fluorescent protein (YFP), we recently observed that deletion of the second RNA recognition motif (RRM2) caused the protein to phase separate and form intranuclear liquid-like droplets. Here, we use fusion constructs of MATR3, TARDBP43 (TDP43) and FUS with YFP or mCherry to examine phase separation and protein colocalization in mouse C2C12 myoblast cells. We observed that the N-terminal 397 amino acids of MATR3 (tagged with a nuclear localization signal and expressed as a fusion protein with YFP) formed droplet-like structures within nuclei. Introduction of the myopathic S85C mutation into NLS-N397 MATR3:YFP, but not ALS mutations F115C or P154S, inhibited droplet formation. Further, we analyzed interactions between variants of MATR3 lacking RRM2 (ΔRRM2) and variants of TDP43 with disabling mutations in its RRM1 domain (deletion or mutation). We observed that MATR3:YFP ΔRRM2 formed droplets that appeared to recruit the TDP43 RRM1 mutants. Further, coexpression of the NLS-397 MATR3:YFP construct with a construct that encodes the prion-like domain of TDBP43 produced intranuclear droplet-like structures containing both proteins. Collectively, our studies show that N-terminal sequences in MATR3 can mediate phase separation into intranuclear droplet-like structures that can recruit TDP43 under conditions of low RNA binding.

RNP-MaP: In-cell analysis of protein interaction networks defines functional hubs in RNA

“…PTBP1, MATR3 and TIA1 proteins are implicated in formation of RNA foci [47][48][49][50] , likely through multivalent interactions between RNAs and proteins 62 , consistent with the highly interactive protein networks we measure in the XIST E region (Fig. 4a).…”

“…XIST E region is critical to maintenance of the silenced X chromosome compartment 33 . Notably, PTBP1, MATR3, and TIA1 each undergo liquid-liquid phase-transitions to form RNA granules [47][48][49][50] , a feature consistent with the formation of an XIST-mediated compartment. PTBP1 and MATR3 interact together on other RNAs as well, creating multi-valent interactions typical of RNA-protein granules 51,52 .The Splicing community includes components known to control splicing (U2AF2, SRSF1, TRA2A, AQR, and ILF3) 53-55 and a chromatin modulator (GRWD1) 56 .…”

Biomolecular condensates in neurodegeneration and cancer

“…Thus, many more condensates are likely to contribute to different diseases. Indeed, altered condensate regulation has been associated with pathologies that are not discussed here, such as SGs in viral infection and myopathy, and Balbiani bodies and infertility …”

Sequestration of TDP-43216-414aggregates by cytoplasmic expression of the proSAAS chaperone

“…The first is the nuage protein DDX4, which forms micron-size spherical "organelles" (Nott et al, 2015). The second protein condensate is a portion of the RNA-binding protein matrin 3 (MATR3) (Gallego-Iradi et al, 2019), implicated in ALS. This protein contains an N-terminal domain involved in the phase separation of MATR3 droplets (Gallego-Iradi et al, 2019).…”

“…The second protein condensate is a portion of the RNA-binding protein matrin 3 (MATR3) (Gallego-Iradi et al, 2019), implicated in ALS. This protein contains an N-terminal domain involved in the phase separation of MATR3 droplets (Gallego-Iradi et al, 2019). A third nuclear protein which clearly forms distinct spheres is the renal protein nephrin (Pak et al, 2016).…”