C2H2-zinc-finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes.

Cys2-His2 zinc-finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs) yet remain largely uncharacterized. Although certain family members, e.g., GTF3A, have been shown to bind both DNA and RNA, the extent to which C2H2-ZNFs interact with-and regulate-RNA-associated processes is not known. Using UV crosslinking and immunoprecipitation (CLIP), we observe that 148 of 150 analyzed C2H2-ZNFs bind directly to RNA in human cells. By integrating CLIP sequencing (CLIP-seq) RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction assays, and transcriptome profiling experiments, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus define a substantially expanded repertoire of C2H2-ZNFs that bind RNA and provide an important resource for elucidating post-transcriptional regulatory programs.

A central chaperone-like role for 14-3-3 proteins in human cells.

14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in Tetrahymena thermophila Reveals an Ancient Network of Chaperones.

Epigenetic information, which can be passed on independently of the DNA sequence, is stored in part in the form of histone posttranslational modifications and specific histone variants. Although complexes necessary for deposition have been identified for canonical and variant histones, information regarding the chromatin assembly pathways outside of the Opisthokonts remains limited. Tetrahymena thermophila, a ciliated protozoan, is particularly suitable to study and unravel the chromatin regulatory layers due to its unique physical separation of chromatin states in the form of two distinct nuclei present within the same cell. Using a functional proteomics pipeline, we carried out affinity purification followed by mass spectrometry of endogenously tagged T. thermophila histones H2A, H2B and variant Hv1.We identified a set of interacting proteins shared among the three analyzed histones that includes the FACT-complex, as well as H2A- or Hv1-specific chaperones. We find that putative subunits of T. thermophila versions of SWR- and INO80-complexes, as well as transcription-related histone chaperone Spt6Tt specifically copurify with Hv1. We also identified importin ß6 and the T. thermophila ortholog of nucleoplasmin 1 (cNpl1Tt) as H2A-H2B interacting partners. Our results further implicate Poly [ADP-ribose] polymerases in histone metabolism. Molecular evolutionary analysis, reciprocal affinity purification coupled to mass spectrometry experiments, and indirect immunofluorescence studies using endogenously tagged Spt16Tt (FACT-complex subunit), cNpl1Tt, and PARP6Tt underscore the validity of our approach and offer mechanistic insights. Our results reveal a highly conserved regulatory network for H2A (Hv1)-H2B concerning their nuclear import and assembly into chromatin.

RACS: rapid analysis of ChIP-Seq data for contig based genomes.

BACKGROUND: Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-Seq) is a widely-used molecular method to investigate the function of chromatin-related proteins by identifying their associated DNA sequences on a genomic scale. ChIP-Seq generates large quantities of data that is difficult to process and analyze, particularly for organisms with a contig-based sequenced genomes that typically have minimal annotation on their associated set of genes other than their associated coordinates primarily predicted by gene finding programs. Poorly annotated genome sequence makes comprehensive analysis of ChIP-Seq data difficult and as such standardized analysis pipelines are lacking. RESULTS: We present a one-stop computational pipeline, "Rapid Analysis of ChIP-Seq data" (RACS), that utilizes traditional High-Performance Computing (HPC) techniques in association with open source tools for processing and analyzing raw ChIP-Seq data. RACS is an open source computational pipeline available from any of the following repositories https://bitbucket.org/mjponce/RACS or https://gitrepos.scinet.utoronto.ca/public/?a=summary&p=RACS . RACS is particularly useful for ChIP-Seq in organisms with contig-based genomes that have poor gene annotation to aid protein function discovery.To test the performance and efficiency of RACS, we analyzed ChIP-Seq data previously published in a model organism Tetrahymena thermophila which has a contig-based genome. We assessed the generality of RACS by analyzing a previously published data set generated using the model organism Oxytricha trifallax, whose genome sequence is also contig-based with poor annotation. CONCLUSIONS: The RACS computational pipeline presented in this report is an efficient and reliable tool to analyze genome-wide raw ChIP-Seq data generated in model organisms with poorly annotated contig-based genome sequence. Because RACS segregates the found read accumulations between genic and intergenic regions, it is particularly efficient for rapid downstream analyses of proteins involved in gene expression.

Molecular evolution of NASP and conserved histone H3/H4 transport pathway.

BACKGROUND: NASP is an essential protein in mammals that functions in histone transport pathways and maintenance of a soluble reservoir of histones H3/H4. NASP has been studied exclusively in Opisthokonta lineages where some functional diversity has been reported. In humans, growing evidence implicates NASP miss-regulation in the development of a variety of cancers. Although a comprehensive phylogenetic analysis is lacking, NASP-family proteins that possess four TPR motifs are thought to be widely distributed across eukaryotes. RESULTS: We characterize the molecular evolution of NASP by systematically identifying putative NASP orthologs across diverse eukaryotic lineages ranging from excavata to those of the crown group. We detect extensive silent divergence at the nucleotide level suggesting the presence of strong purifying selection acting at the protein level. We also observe a selection bias for high frequencies of acidic residues which we hypothesize is a consequence of their critical function(s), further indicating the role of functional constraints operating on NASP evolution. Our data indicate that TPR1 and TPR4 constitute the most rapidly evolving functional units of NASP and may account for the functional diversity observed among well characterized family members. We also show that NASP paralogs in ray-finned fish have different genomic environments with clear differences in their GC content and have undergone significant changes at the protein level suggesting functional diversification. CONCLUSION: We draw four main conclusions from this study. First, wide distribution of NASP throughout eukaryotes suggests that it was likely present in the last eukaryotic common ancestor (LECA) possibly as an important innovation in the transport of H3/H4. Second, strong purifying selection operating at the protein level has influenced the nucleotide composition of NASP genes. Further, we show that selection has acted to maintain a high frequency of functionally relevant acidic amino acids in the region that interrupts TPR2. Third, functional diversity reported among several well characterized NASP family members can be explained in terms of quickly evolving TPR1 and TPR4 motifs. Fourth, NASP fish specific paralogs have significantly diverged at the protein level with NASP2 acquiring a NNR domain.

Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48.

BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation.

BACKGROUND: Eukaryotic cells can rapidly adjust their transcriptional profile in response to molecular needs. Such dynamic regulation is, in part, achieved through epigenetic modifications and selective incorporation of histone variants into chromatin. H3.3 is the ancestral H3 variant with key roles in regulating chromatin states and transcription. Although H3.3 has been well studied in metazoans, information regarding the assembly of H3.3 onto chromatin and its possible role in transcription regulation remain poorly documented outside of Opisthokonts. RESULTS: We used the nuclear dimorphic ciliate protozoan, Tetrahymena thermophila, to investigate the dynamics of H3 variant function in evolutionarily divergent eukaryotes. Functional proteomics and immunofluorescence analyses of H3.1 and H3.3 revealed a highly conserved role for Nrp1 and Asf1 histone chaperones in nuclear influx of histones. Cac2, a putative subunit of H3.1 deposition complex CAF1, is not required for growth, whereas the expression of the putative ortholog of the H3.3-specific chaperone Hir1 is essential in Tetrahymena. Our results indicate that Cac2 and Hir1 have distinct localization patterns during different stages of the Tetrahymena life cycle and suggest that Cac2 might be dispensable for chromatin assembly. ChIP-seq experiments in growing Tetrahymena show H3.3 enrichment over the promoters, gene bodies, and transcription termination sites of highly transcribed genes. H3.3 knockout followed by RNA-seq reveals large-scale transcriptional alterations in functionally important genes. CONCLUSION: Our results provide an evolutionary perspective on H3.3's conserved role in maintaining the transcriptional landscape of cells and on the emergence of specialized chromatin assembly pathways.

RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific.

Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)-proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.

SARS-CoV-2 nucleocapsid protein binds host mRNAs and attenuates stress granules to impair host stress response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is essential for viral replication, making it a promising target for antiviral drug and vaccine development. SARS-CoV-2 infected patients exhibit an uncoordinated immune response; however, the underlying mechanistic details of this imbalance remain obscure. Here, starting from a functional proteomics workflow, we cataloged the protein-protein interactions of SARS-CoV-2 proteins, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N localizes to stress granules and sequesters G3BPs away from their typical interaction partners, thus attenuating stress granule formation. We found that N binds directly to host mRNAs in cells, with a preference for 3' UTRs, and modulates target mRNA stability. We show that the N protein rewires the G3BP1 mRNA-binding profile and suppresses the physiological stress response of host cells, which may explain the imbalanced immune response observed in SARS-CoV-2 infected patients.

Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila.

We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan Tetrahymena thermophila. The FZZ epitope tag contains 2 protein A moieties (ZZ) and a 3xFLAG separated by a TEV cleavage site, which can also be employed in tandem affinity purification. This protocol is versatile and is suitable to use for other common epitope tags and can be adapted for other ciliates. For complete details on the use and execution of this protocol, please refer to Garg et al. (2019).

Exploring the Histone Acetylation Cycle in the Protozoan Model Tetrahymena thermophila.

The eukaryotic histone acetylation cycle is composed of three classes of proteins, histone acetyltransferases (HATs) that add acetyl groups to lysine amino acids, bromodomain (BRD) containing proteins that are one of the most characterized of several protein domains that recognize acetyl-lysine (Kac) and effect downstream function, and histone deacetylases (HDACs) that catalyze the reverse reaction. Dysfunction of selected proteins of these three classes is associated with human disease such as cancer. Additionally, the HATs, BRDs, and HDACs of fungi and parasitic protozoa present potential drug targets. Despite their importance, the function and mechanisms of HATs, BRDs, and HDACs and how they relate to chromatin remodeling (CR) remain incompletely understood. Tetrahymena thermophila (Tt) provides a highly tractable single-celled free-living protozoan model for studying histone acetylation, featuring a massively acetylated somatic genome, a property that was exploited in the identification of the first nuclear/type A HAT Gcn5 in the 1990s. Since then, Tetrahymena remains an under-explored model for the molecular analysis of HATs, BRDs, and HDACs. Studies of HATs, BRDs, and HDACs in Tetrahymena have the potential to reveal the function of HATs and BRDs relevant to both fundamental eukaryotic biology and to the study of disease mechanisms in parasitic protozoa.

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

Nucleus-specific linker histones Hho1 and Mlh1 form distinct protein interactions during growth, starvation and development in Tetrahymena thermophila.

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle.

Functional Proteomics of Nuclear Proteins in Tetrahymena thermophila: A Review.

Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena, to address important questions and better understand nuclear processes including chromatin biology of eukaryotes.