InteractiveFly: GeneBrief

DISCO Interacting Protein 1: Biological Overview | References

BIOLOGICAL OVERVIEW

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. This study report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, this study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila. Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. The study shows that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis (Wong, 2017).

Recent studies have uncovered a class of stable intronic sequence RNAs (sisRNAs) that are derived from the introns post splicing. sisRNAs are present in various organisms such as viruses, yeast, Drosophila, Xenopus, and mammals. Studies in Drosophila and mammalian cells suggest that sisRNAs function in regulating the expression of their parental genes (host genes where they are derived from) via positive or negative feedback loops (Pek, 2015; Zhang, 2013; Zheng, 2015). In yeast, sisRNAs are involved in promoting robustness in response to stress, while in Drosophila, sisRNAs have been shown to be important for embryonic development (Tay, 2017). However, very little is understood about the biological functions of sisRNAs in terms of regulating cellular processes such as differentiation, proliferation, and cell death (Wong, 2017).

The Drosophila genome encodes for several double-stranded RNA (dsRNA) binding proteins that localize to the nucleus. Most of them have been found to regulate specific RNA-mediated processes such as RNA editing, X chromosome activation, and miRNA biogenesis. The Disco-interacting protein 1 (DIP1) is a relatively less characterized dsRNA binding protein that has been implicated in anti-viral defense and localizes to the nucleus as speckles. Otherwise, not much is known about the biological processes regulated by DIP1 (Catanese, 2010; Cantanese, 2011; DeSousa, 2003; Zhang, 2015; Wong, 2017 and references therein).

This paper show that the regulation of a Drosophila sisRNA sisR-1 by DIP1 is important for keeping female germline stem cell homeostasis in place. DIP1 is shown to regulate INE-1 sisRNAs and localizes to a previously undescribed nuclear body around the fourth chromosomes, called the satellite body. The regulation of sisR-1, which is not on the fourth chromosome, by DIP1 presumably does not occur in the satellite bodies (Wong, 2017).

The results reveal the importance of the regulation of sisRNA activity/expression in GSC-niche occupancy. It is proposed that the sisR-1 axis maintains GSCs in the niche, however, uncontrolled accumulation of sisR-1 due to its unusual stability can lead to increase number of GSCs at the niche. DIP1 in turn limits the build-up of sisR-1 to maintain ~2 GSCs per niche. GSC-niche occupancy is highly regulated by homeostatic mechanisms via negative feedback loops at the cellular and molecular levels. Misregulation of the niche may pose a problem as it allows for a greater chance of GSCs to accumulate mutations that may lead to tumor formation. On the other hand, mechanisms that promote GSC-niche occupancy may be important to facilitate the replenishment of GSCs during aging. Understanding the control of stem cell-niche occupancy will provide important insights to reproduction, cancer, and regenerative medicine (Wong, 2017).

In a large-scale RNAi screen for genes that regulate GSC self-renewal and differentiation, rga was identified as a gene required for GSC differentiation (Pek, 2015). How rga regulates GSC self-renewal is currently unknown but the current data suggest that GSC-niche adhesion and Mei-P26 are involved. The rga gene encodes for the NOT2 protein in the CCR4 deadenylase complex. Surprisingly, studies have shown that other components of the CCR4 complex such as CCR4, Not1, and Not3 function in promoting GSC maintenance. Interestingly, Twin has been proposed to function with distinct partners to mediate different effects on GSC fates. This suggests that other components such as CCR4 can also have additional functions outside the CCR4-NOT deadenylase complex in mediating GSC maintenance, thus affecting GSCs in opposite ways to Rga (Wong, 2017).

This study puts forward a proposed model for sisR-1-mediated silencing. It is hypothesized that folded sisR-1 harboring a 3' tail may form a ribonucleoprotein complex, which confers its stability, and allows scanning for its target via its 3' tail. Binding of the 3' tail to the target may promote local unwinding of sisR-1 as the 3' end of ASTR invades to form a more stable 76 nt duplex. This study shows that, in principle, it is possible to design a chimeric sisRNA to target a long ncRNA of interest such as rox1. In future, sisRNA can be potentially developed as tools to regulate nuclear RNAs of interest. Clearly, the efficiency and specificity of sisRNA-mediated silencing need to be optimized. Because sisRNA-mediated target degradation requires a more extensive base-pairing between sisRNA and the target, the chances of off-target effects ought to be lower than siRNAs and antisense oligonucleotides. In broader terms, this study provides a paradigm, which encourages exploration of whether other sisRNAs or ncRNAs utilize a similar silencing strategy as sisR-1 (Wong, 2017).

This study describes a nuclear body (named satellite body) that associates with the fourth chromosomes. Satellite body adds to an existing group of nuclear bodies (nucleolus, HLB, and pearl) that associate with specific genomic loci. It is generally believed that formation of such nuclear bodies correlates with a high concentration of RNA transcribed from the tandemly repeated gene loci. The formation of satellite bodies around the fourth chromosomes probably reflects a high concentration of DIP1 in regulating INE-1 sisRNAs transcribed there. The formation of satellite bodies may be promoted by the high concentration of INE-1 sisRNAs transcribed on the fourth chromosomes, and may facilitate the decay of INE-1 sisRNAs . It is speculated that in the nucleoplasm, DIP1 that does not form observable satellite bodies is sufficient to regulate sisRNAs such as sisR-1 transcribed from other chromosomes. Since DIP1 is a dsRNA binding protein, it may bind to mature sisRNAs to destabilize them. It may do so by recruiting RNA degradation factors (such as nuclear exosomes) or introducing RNA modification to 'mark' sisRNAs for degradation. In future, it will be important to identify more components of the satellite bodies and their dynamics during differentiation and in response to stimuli in order to better understand the molecular mechanism of sisRNA metabolism (Wong, 2017).

DIP1 plays an antiviral role against DCV infection in Drosophila melanogaster

Disconnected Interacting Protein 1 (DIP1) is a dsRNA-binding protein that participates in a wide range of cellular processes. Whether DIP1 is involved in innate immunity remains unclear. DIP1 has been found to play an antiviral role in S2 cells. Its antiviral action is specific for DCV infection and not for DXV infection. dip1 mutant flies are hypersensitive to DCV infection. The increased mortality in dip1 mutant flies is associated with the accumulation of DCV positive-stranded RNAs in vivo. This study demonstrated that dip1 is a novel antiviral gene that restricts DCV replication in vitro and in vivo (Zhang, 2015).

Disconnected Interacting Protein 1 binds with high affinity to pre-tRNA and ADAT

Disconnected Interacting Protein 1 (DIP1), a member of the double-stranded RNA-binding protein family based on amino acid sequence, was shown previously to form complexes with multiple transcription factors in Drosophila melanogaster. To explore this protein further, sedimentation equilibrium experiments were undertaken that demonstrate that DIP1-c (longest isoform of DIP1) is a dimer in solution, a characteristic common to other members of the dsRNA-binding protein family. The closest sequence identity for DIP1 is found within the dsRBD sequences of RNA editase enzymes. Consistent with this role, this study demonstrates binding of DIP1-c to a potential physiological RNA target: pre-tRNA. In addition, DIP1-c was shown to interact with ADAT, a tRNA deaminase that presumably modifies pre-tRNAs. From these data, it is hypothesized that DIP1 may serve an integrator role by binding its dsRNA ligand and recruiting protein partners for the appropriate metabolism of the bound RNA (Catanese, 2011).

High affinity, dsRNA binding by Disconnected interacting protein 1

Disconnected interacting protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. This study shows that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax (Catanese, 2010).

Characteristics of the structural organization of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flamSS (SS) and flamMS (MS), characterized by a high transposition rate of retrotransposon gypsy ( mdg4), flampy + (P) carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco +. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flamSS, flamMS, flampy +(P), and flamenco+ considerably differ from one another in the structure of DIP1. Strains flamss and flamMS have no Dral restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The Dral gene of strainsflamSS andflamMS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tcl/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIPI and, probably, neighboring genes. In strains flamenco+ and flampy + (P), the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome (Nefedova, 2007).

Hox transcription factor Ultrabithorax Ib physically and genetically interacts with Disconnected interacting protein 1, a double-stranded RNA-binding protein

Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx). The Ubx.DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated (Bondos, 2004).

Identification of a cDNA clone encoding DIP1-binding protein in Drosophila melanogaster

The Drosophila melanogaster L27a gene encodes a ribosomal protein which is a member of the L15 family of ribosomal proteins. D.m. L27a is closely related to the mammalian protein that has been found differentially expressed in lung cancer tissues and therefore could be involved in the control of cell proliferation such as the ribosomal protein S6. This work elucidates the role of DIP1 which is a novel protein that was found in Drosophila. A two-hybrid system assay was performed and the L27a protein was identified as an interactor of DIP1. The interaction was then validated by in vitro binding assays. DIP1, similar to other nuclear proteins in eukaryotes, is localized to the nuclear periphery and chromatin domain in all nuclei, but disappears at the metaphase. It is possible that in D.m. L27a protein, via interaction with DIP1, could be involved in protein synthesis as well as in cell cycle regulation (De Felice, 2004).

A novel double-stranded RNA-binding protein, disco interacting protein 1 (DIP1), contributes to cell fate decisions during Drosophila development

This study reports the identification of the Disco Interacting Protein 1 (DIP1) gene isolated in a yeast interaction trap screen using the zinc finger protein Disconnected (Disco) as a bait. DIP1 encodes a protein containing two double-stranded RNA binding domains (dsRBD). Consistent with the presence of dsRBD, DIP1 binds dsRNA or structured RNAs in Northwestern assays. DIP1 is found in nuclear subdomains resembling speckles known to accumulate transcription and splicing factors. In early embryos, nuclear localization of DIP1 protein coincides with the onset of zygotic gene expression. Later in development DIP1 expression is decreased in dividing cells in different tissues. Overexpression of DIP1 in the eye-antennal imaginal disc, early in embryonic and larval development, causes the formation of supernumerary structures in the head capsule. A role for DIP1 in epigenetic mechanisms that lead to the establishment and/or maintenance of cell fate specification is discussed (DeSousa, 2003).

Search PubMed for articles about Drosophila DIP1

Bondos, S.E., Catanese, D.J., Tan, X.X., Bicknell, A., Li, K., Matthews, K.S. (2004). Hox transcription factor ultrabithorax Ib physically and genetically interacts with Disconnected interacting protein 1, a double-stranded RNA-binding protein. J. Biol. Chem. 279(25): 26433-26444. PubMed ID: 15039447

Catanese, D. J., Jr. and Matthews, K. S. (2010). High affinity, dsRNA binding by Disconnected interacting protein 1. Biochem Biophys Res Commun 399(2): 186-191. PubMed ID: 20643095

Catanese, D. J., Jr. and Matthews, K. S. (2011). Disconnected Interacting Protein 1 binds with high affinity to pre-tRNA and ADAT. Biochem Biophys Res Commun 414(3): 506-511. PubMed ID: 21971547

De Felice, B., Ciarmiello, L. F. and Wilson, R. R. (2004). Identification of a cDNA clone encoding DIP1-binding protein in Drosophila melanogaster. Mol Biol Rep 31(3): 165-169. PubMed ID: 15560371

DeSousa, D., Mukhopadhyay, M., Pelka, P., Zhao, X., Dey, B. K., Robert, V., Pelisson, A., Bucheton, A. and Campos, A. R. (2003). A novel double-stranded RNA-binding protein, disco interacting protein 1 (DIP1), contributes to cell fate decisions during Drosophila development. J Biol Chem 278(39): 38040-38050. PubMed ID: 12829713

Nefedova, L. N., Romanova, N. I. and Kim, A. I. (2007). [Characteristics of the structural organization of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene]. Genetika 43(1): 70-77. PubMed ID: 17333941

Pek, J. W., Osman, I., Tay, M. L. & Zheng, R. T (2015). Stable intronic sequence RNAs have possible regulatory roles in Drosophila melanogaster. J. Cell Biol. 211, 243–251. PubMed ID: 26504165

Wong, J. T., Akhbar, F., Ng, A. Y. E., Tay, M. L., Loi, G. J. E. and Pek, J. W. (2017). DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1. Nat Commun 8(1): 759. PubMed ID: 28970471

Tay, M. L. and Pek, J. W. (2017). Maternally Inherited Stable Intronic Sequence RNA Triggers a Self-Reinforcing Feedback Loop during Development. Curr Biol 27(7): 1062-1067. PubMed ID: 28343963

Zhang, Q., Zhang, L., Gao, X., Qi, S., Chang, Z. and Wu, Q. (2015). DIP1 plays an antiviral role against DCV infection in Drosophila melanogaster. Biochem Biophys Res Commun 460(2): 222-226. PubMed ID: 25770426

Zhang, Y., Zhang, X. O., Chen, T., Xiang, J. F., Yin, Q. F., Xing, Y. H., Zhu, S., Yang, L. and Chen, L. L. (2013). Circular intronic long noncoding RNAs. Mol Cell 51(6): 792-806. PubMed ID: 24035497

Zheng, S., Vuong, B. Q., Vaidyanathan, B., Lin, J. Y., Huang, F. T. and Chaudhuri, J. (2015). Non-coding RNA Generated following Lariat Debranching Mediates Targeting of AID to DNA. Cell 161(4): 762-773. PubMed ID: 25957684

date revised: 22 November 2022

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