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

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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. 3. 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 (16). 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. A manuscript of this work is under review.