Investigating the roles of RNA-binding proteins Nocte and PRRC2 in mRNA translation and age-related neurodegeneration using Drosophila and mouse models

National Institute On Aging

Investigators

Linked publications & trials

Abstract

RNA-binding proteins play important roles in aging and age-related neurodegeneration by regulating mRNA translation, stability and RNA granule dynamics. Wang group has long-term research interests on Topoisomerase 3b (Top3b), the main topoisomerase that can bind RNA and catalyze the release of RNA topological stress. Top3b mutation has been linked to schizophrenia and autism. Wang group has studied the functions of Top3b and its binding partner TDRD3 in mRNA translation in cell line, Drosophila and mouse models. The powerful genetic tools and less gene redundancy in Drosophila studies can help us to discover in vivo functions of proteins and study their mechanisms efficiently. We used IP-Mass Spec to identify Top3b/TDRD3 interacting proteins in Drosophila, mouse and human cells. We found that Drosophila protein Nocte and its mammalian ortholog PRRC2A are strong Top3b/TDRD3 interacting proteins. Previous studies show that a partial mutation of nocte in Drosophila leads to defects in temperature compensation of the circadian clock. Studies in mouse and human cells show that 1) PRRC2A and PRRC2C are stress granule proteins; 2) PRRC2A binds m6A modified mRNAs and plays a role in neural development; 3) Polymorphism of PRRC2A has been associated with several diseases, including age-at-onset diabetes mellitus. However, the molecular functions of this family of proteins are still largely unknown. We have investigated the functions of Nocte in Drosophila and have made following findings: 1) We generated nocte knockouts by CRISPR and found that nocte-KO flies die at larval-pupal stages. Specific knockdown of nocte by RNAi in fly eyes induces rough and small eye phenotypes. 2) RNA-seq, immunostaining, qRT-PCR and RIP-qPCR results show that Nocte is necessary and sufficient to promote the translation of glass mRNA which encodes a transcription factor important for Drosophila eye development. 3) With reporter assays and CRISPR edited alleles, we demonstrated that Nocte counteracts the suppression effects of the upstream ORF (uORF) in glass 5UTR to maintain glass translation. 4) We tested genetic interactions between Nocte and several Drosophila neurodegeneration models, and found that Nocte has strong genetic interactions with Huntington and ALS/FTD models induced by expression of amino acid repeats. With these results, we plan to further study the mechanisms of how Nocte/PRRC2 regulates mRNA translation and pathogenesis of neurodegeneration in Drosophila and cell lines, and collaborate with Dr. Huaibin Cais group in LNG and Dr. Nigel Greigs group in TGB. Specific Aims: Aim1. Studying functions of Nocte/PRRC2A in mRNA translation. Our current studies indicate that Nocte positively regulates translation of glass mRNA by counteracting the suppression effects of the longest uORF in glass 5UTR. We propose two models of Noctes function on glass translation: re-initiation and ribosome recruitment. In ribosome recruitment model, Nocte may bind Internal Ribosome Entry Sites (IRES) in glass 5UTR to recruit translation re-initiation complex. We will use a IRES reporter to test this model (11). We will also use CLIP to determine Noctes binding sites on glass mRNA. Furthermore, we will study Noctes roles on translation at genome-wide scale by Ribo-seq and puromycin-associated nascent chain proteomics (PUNCH-P). We have generated PRRC2A-KO Hela cell lines and tested PRRC2C-RNAi efficiency in Hela cells. We plan to do RNA-seq and Ribo-seq to check whether PRRC2 also regulates mRNA translation by using the knockout and knockdown cell lines. Aim2. Investigating functions of Nocte/PRRC2 in neurodegeneration caused by amino acid repeats. Our results of genetic studies indicate that Nocte enhances the phenotypes induced by polyQ in HD model but suppresses the phenotypes induced by polyGR and polyPR in ALS/FTD model. We will investigate whether the protein levels of the pathogenic amino acid repeats are regulated by Nocte and whether Nocte regulates the dynamics of aggregates and/or normal RNA granules by immunostaining with markers. The plasmids for the overexpression of polyQ and GGGGCC repeats that encode polyGR and polyPR in mammalian cells had been used in previous studies. The overexpression of these repeats leads to cell apoptosis, cell cycle arrest, protein aggregate formation and changes of stress granule dynamics. We will request these plasmids and test whether loss and overexpression of PRRC2A and PRRC2C can modify the phenotypes caused by the overexpression of the repeats in cell lines. Aim3. Examining the functions of PRRC2A in mouse Huntington and ALS/FTD models. We are interested in whether PRRC2A have interactions with HD and ALS/FTD in mouse models. Huntington (R6/2) and ALS/FTD (Tg(C9orf72_3)) mouse lines are available in Jackson lab. PRRC2A-KO mice were also generated recently. We will request these mice and generate double-mutant progenies to study the genetic interactions. We will collaborate with Dr. Cais group to analyze the pathogenesis in neurons, and with Dr. Greigs group to study the behavior changes of these mice. Results: 1.Nocte is necessary and sufficient for translation of glass mRNA in developing Drosophila eye. We generated nocte-KO flies with CRISPR, and found that they died during development and could not survive to adult stage. Flies with specific nocte knockdown by RNAi in developing eyes can survive to adult stages and show small and rough eye phenotypes by comparing with WT eye. We found that the expression of Glass, a transcription factor with important functions in eye development, is largely decreased by nocte-RNAi in developing eye tissues at larval stage. The RNA-seq results show that glass mRNA levels are similar in WT and nocte-RNAi eyes, which is confirmed by qRT-PCR. RNA-immunoprecipitation (RIP) with Flag.Nocte shows that Nocte has much stronger binding on glass mRNA than a housekeeping mRNA. All these data suggest that Nocte binds glass mRNA and regulates its translation. 2. Nocte may regulate glass translation by counteracting the suppression effects of uORF To test whether the regulation of Nocte depends on uORFs, we took the advantage of a CRISPR-edited glass allele 5nt::glass generated by Sprecher lab. 5 base pairs are deleted in the glass 5UTR in 5nt::glass allele, which leads to the fusion of uORF2 and glass main ORF (Fig. 3A). As a result, the CDS of 5nt::glass does not have the translation inhibition by a long uORF. In WT background, the protein levels of Glass from WT glass and 5nt::glass do not show significant differences. Notably, nocte-RNAi strongly suppress the Glass protein encoded by WT glass but not 5nt::glass (Fig. 3B-C), suggesting that the regulation of Nocte on glass translation depends on uORF2 of glass mRNA. A manuscript has been published (Zhang, et al., NAR 2024). 3. Nocte cooperates with eIF3d and eIF4G2 to regulate uORF-dependent translation. About half of mammalian mRNAs contain uORFs, which are critical for translation of stress response genes, including ATF4. uORF-dependent translation has been associated with age-associated disorders, such as AD and Hungtington disease (HD). The findings by our group and others that Nocte and its mammalian orthologs enhance uORF-dependent translation indicate that they play a conserved role in this process. We therefore continued to explore the underlying mechanism. Our current evidence supports a model that Nocte works with eIF3d/eIF4G2 alternative translation initiation machinery to regulate the process. 4. Genetic interactions between Nocte and neurodegeneration caused by expression of amino acid repeats. There are established fly neurodegeneration models caused by expression of nucleotide repeats in their eyes. The neurodegeneration defects are reflected by eye pigment loss and roughness. Comparing to the GMR-Gal4 only as control, overexpression or knockdown of Nocte does not cause obvious eye defects. Overexpression of Huntingtin with poly120Q induces partial pigment loss in the eye. Overexpression of Huntingtin with poly120Q induces partial pigment loss in the eye. Overexpression of Nocte strongly enhances the phenotypes, while Nocte-RNAi significantly suppresses the defects. For ALS/FTD models, Nocte-RNAi enhances the pigment loss/roughness phenotypes induced by expression of poly36GGGGCC that encodes poly36GR and poly36PR or expression of just the amino acid repeats, poly36GR or poly36PR; while overexpression of Nocte does not change or make mild suppression. 5. Targeting Prion-like RBPs to suppress neurodegeneration mediated by Huntington Disease protein. Huntington disease (HD) is caused by CAG repeat expansion in Huntingtin (HTT) gene. The polyQ peptide expressed from the expansion can fold into prion-like stacks to form protein aggregates, whose sizes and numbers are increased by age and positively correlate with the disease severity. However, therapeutics to remove these aggregates and suppress HTT-induced neurotoxicity are not yet available. Over-expression of some prion-like endogenous proteins have been found to suppress HTT-induced toxicity in yeast. Our studies on the functions of prion-like RBPs in HTT-induced neurodegeneration were inspired by the strong genetic interactions between HTT and Nocte, the RBP which we are working on(Zhang et al., NAR 2024). Nocte and its mammalian homologs (PRRC2s) have several prion-like regions (with high prion scores), and co-expression of Nocte with HTT induces strong synergistic eye defects and formation of co-aggregates. Interestingly, Nocte-depletion by RNAi partially suppresses HTT-induced eye defects, suggesting endogenous Nocte promotes HTT neurotoxicity. The Nocte-HTT data prompted us to systematically investigate the functions of all prion-like RBPs in HTT-induced neurodegeneration in Drosophila eyes. Using prion scores, we identified 124 Drosophila prion-like RBPs that have human homologs. We then performed RNAi depletion (knockdown, KD) and over-expression (OE) screens to determine which ones can suppress or enhance HTT-induced phenotypes. Our RNAi screens identified 18 RBPs whose depletion suppresses, and 18 other RBPs whose depletion enhances, the HTT-induced eye defects. By combining the OE and RNAi screens, we identified two genes (including Rox8) behaving similarly as nocte: enhancement of HTT toxicity by OE and suppression by RNAi, while another two genes (including Atx2) show enhancement by both OE and RNAi. To explain these differences, we hypothesize that the prion-like RPBs that interact with HTT can be classified into at least two functional categories: Helpers (such as Rox8 and Nocte) that help HTT to form the aggregates by recruiting other RBPs; and Targets (such as Atx2) that are specific RBPs recruited to the aggregates by Helpers, so that they lost their normal localization and function, leading to neurotoxicity. This hypothesis predicts that either KD of Helpers or restoring Targets by deleting their prion-like regions should suppress HTT-mediated toxicity. Our follow-up studies support this hypothesis. Furthermore, human homologs of Nocte (PRRC2s) and Rox8 (TIA1 and TIAL1) can form co-aggregates with HTT in a human cell line; and depletion of these homologs can reduce HTT-induced apoptosis. Previous studies show that reduction of TIA1 suppresses Tau-mediated neurodegeneration in mouse. Together, these data suggest that prion-like RBPs can be targeted to suppress HTT-mediated neurodegeneration.