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Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. The protein Fused in Sarcoma (FUS) undergoes LLPS and mutations in FUS have been causally linked to the motor neuron disease Amyotrophic Lateral Sclerosis (ALS-FUS). LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. However, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. Hence, we developed a method allowing for the purification of LLPS FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome, depending on its biophysical state. While non-LLPS FUS interacts mainly with factors involved in pre-mRNA processing, LLPS FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, also mitochondrial factors are strongly enriched with LLPS FUS, providing a potential explanation for the observed changes in mitochondrial gene expression in mouse models of ALS-FUS. In summary, we present a methodology to investigate the interactomes of phase separating proteins and provide evidence that LLPS shapes the FUS interactome with implications for function and disease.

Accumulation of DNA damage in neurons is largely described in aging process and neurodegenerative diseases. Recently, RNA-binding proteins (RBPs) have emerged as important effectors of the cellular DNA damage response (DDR). The intrinsically disordered RBP FUS has been shown to play a role in multiple cellular functions, including mRNA transcription, splicing and transport, and in DDR. Besides that, FUS, together with some other RBPs, has been shown to undergo liquid-liquid phase separation (LLPS) under physiological conditions, but its precise molecular functions in DDR and the role of its LLPS remain poorly understood. The aims of this study are to: i) characterise the molecular functions of FUS in the DDR, ii) assess whether a proper repair of double strand breaks (DSB) is dependent on the LLPS of FUS. Using gene-edited HeLa cell lines, we showed that knock-out of FUS (FUS-KO) sensitised cells to genotoxic stress, impaired DSB repair, and caused changes in DDR pathways. At the molecular level, we found that FUS-KO impaired the recruitment of apical effectors of the DDR to laser-induced DNA damage sites. FUS itself is recruited within seconds to DSBs and is required for the retention of the apical DNA DSBs sensor KU. Mass spectrometry analysis revealed that FUS co-purifies with RBP implicated in DDR, including SFPQ. We demonstrate that LLPS occurs at DNA damage foci and is required for the efficient recruitment of key DDR factors. Finally, LLPS-deficient FUS variants impair the recruitment of KU and SFPQ. Overall, our findings establish for the first time that FUS-dependent LLPS contributes to the activation of the DDR and to the recruitment of DDR proteins at sites of DNA damage.

Accumulation of DNA damage in neurons is largely described in aging process and neurodegenerative diseases. Recently, RNA-binding proteins (RBPs) have emerged as important effectors of the cellular DNA damage response (DDR). The intrinsically disordered RBP FUS has been shown to play a role in multiple cellular functions, including mRNA transcription, splicing and transport, and in DDR. Besides that, FUS, together with some other RBPs, has been shown to undergo liquid-liquid phase separation (LLPS) under physiological conditions, but its precise molecular functions in DDR and the role of its LLPS remain poorly understood. The aims of this study are to: i) characterise the molecular functions of FUS in the DDR, ii) assess whether a proper repair of double strand breaks (DSB) is dependent on the LLPS of FUS. Using gene-edited HeLa cell lines, we showed that knock-out of FUS (FUS-KO) sensitised cells to genotoxic stress, impaired DSB repair, and caused changes in DDR pathways. At the molecular level, we found that FUS-KO impaired the recruitment of apical effectors of the DDR to laser-induced DNA damage sites. FUS itself is recruited within seconds to DSBs and is required for the retention of the apical DNA DSBs sensor KU. Mass spectrometry analysis revealed that FUS co-purifies with RBP implicated in DDR, including SFPQ. We demonstrate that LLPS occurs at DNA damage foci and is required for the efficient recruitment of key DDR factors. Finally, LLPS-deficient FUS variants impair the recruitment of KU and SFPQ. Overall, our findings establish for the first time that FUS-dependent LLPS contributes to the activation of the DDR and to the recruitment of DDR proteins at sites of DNA damage.

Background. Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. Methods. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 +/- 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Results. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Conclusions. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs.

Background. Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. Methods. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 +/- 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Results. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Conclusions. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs.

Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre-mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron-containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS-associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.