RNA Gene Regulation Research Articles

Long non-coding RNA and epigenetic gene regulation of KSHV.

Kaposi’s sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is a γ-herpesvirus linked to Kaposi’s sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL or body-cavity B-lymphoma [BCBL]) and a subset of Multicentric Castleman’s Disease. During lytic growth, pervasive viral transcription generating a variety of transcripts with uncertain protein-coding potential has been described on a genome-wide scale in β- and γ-herpesviruses. One class of such RNAs is called long non-coding RNAs (lncRNAs). KSHV encodes a viral lncRNA known as polyadenylated nuclear RNA (PAN RNA), a copious early gene product. PAN RNA has been implicated in KSHV gene expression, replication, and immune modulation. PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program. Latent KSHV episomes are coated with viral latency-associated nuclear antigen (LANA). LANA rapidly dissociates from episomes during reactivation. Here we review recent studies suggesting that PAN RNA may function as a viral lncRNA, including a role in the facilitation of LANA-episomal dissociation during lytic replication.

RNA Biogenesis: Mechanism and Evolution of RNase III

RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer are representative members of the family. The bacterial enzyme possesses a single RNase III domain (RIIID) followed by a dsRNA-binding domain (dsRBD); Rnt1p is defined by the presence of an N-terminal domain (NTD), a RIIID, and a dsRBD; Drosha contains N-terminal P-rich and RS-rich domains followed by two RIIIDs and a dsRBD; and Dicer possesses N-terminal helicase, DUF283, and PAZ domains followed by two RIIIDs and a dsRBD. It is the N-terminal extension beyond the RIIID that distinguishes eukaryotic RNase IIIs from the bacterial enzyme. My lab has been studying the structure and mechanism of RNase III enzymes since 1996. We have reported a total of eleven crystal structures of bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex (Gan et al., Cell, 124:355-366, 2006), and thereby well characterized the mechanism of action for the bacterial enzyme (Court et al., Annu Rev Genet, 47:405-431, 2013). We have also determined the crystal structure of yeast Rnt1p post-cleavage complex, the first structure of a eukaryotic RNase III complexed with RNA in a catalytically meaningful manner (Liang et al., Molecular Cell, 54:431-444, 2014, featured article on the issue cover). Strikingly, the NTD and dsRBD of Rnt1p function as two rulers for substrate selection. This unusual mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs, including Drosha and Dicer.

Structure of a Eukaryotic RNase III Postcleavage Complex Reveals a Double-Ruler Mechanism for Substrate Selection

Ribonuclease III (RNase III) enzymes are a family of double-stranded RNA (dsRNA)-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III enzymes have been well characterized, but how eukaryotic RNase IIIs work isless clear. Here, we describe the structure of the Saccharomyces cerevisiae RNase III (Rnt1p) postcleavage complex and explain why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of the Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTDs) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs.

Tertiary structure mapping of the pri-miRNA miR-17~92.

The understanding of RNA in regulating gene expression has exploded over the past 15 years. MicroRNAs (miRNAs) have vastly expanded the role of RNA in gene regulation beyond spliceosomal, ribosomal, and messenger RNAs. Approximately one half of miRNAs are polycistronic, where two or more miRNAs are encoded on a single pri-miRNA transcript, termed a miRNA cluster. The six miRNAs of the miR-17~92 cluster are contained within a ~800 nucleotide region within intron 3 of the cl13orf25 ~7 kb pri-miRNA transcript. We recently reported on the tertiary structured domain of miR-17~92 and its role in modulating miRNA biogenesis. The key finding was that the cluster structure explained the differential processing of the miRNA hairpins by Drosha. This work demonstrated the need to consider pri-miRNA tertiary structure in miRNA biogenesis. Since biochemical structure probing is typically performed on relatively short RNAs (≤200 nucleotides), we had to adapt these methodologies for application on large RNAs (~800 nucleotide miR-17~92 pri-miRNA). We present here our adaptation of a protection footprinting method using ribonucleases to probe the structure of the ~800 nucleotide miR-17~92 pri-miRNA. We outline the technical difficulties involved in probing large RNAs and data visualization using denaturing polyacrylamide gel electrophoresis and how we adapted the existing approaches to probe large RNAs. The methodology outlined here is generally applicable to large RNAs including long noncoding RNAs (lncRNA).

Noncoding RNAs in cardiovascular biology and disease.

The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1 The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). …

Mammalian 5′-Capped MicroRNA Precursors that Generate a Single MicroRNA

MicroRNAs (miRNAs) are short RNA gene regulators typically produced from primary transcripts that arecleaved by the nuclear microprocessor complex, with the resulting precursor miRNA hairpins exported by exportin 5 and processed by cytoplasmic Dicer toyield two (5p and 3p) miRNAs. Here, we documentmicroprocessor-independent 7-methylguanosine (m(7)G)-capped pre-miRNAs, whose 5' ends coincide with transcription start sites and 3' ends are most likely generated by transcription termination. By establishing a small RNA Cap-seq method that employs the cap-binding protein eIF4E, we identified a group of murine m(7)G-capped pre-miRNAs genome wide. The m(7)G-capped pre-miRNAs are exported via the PHAX-exportin 1 pathway. After Dicer cleavage, only the 3p-miRNA is efficiently loaded onto Argonaute to form a functional microRNP. Thisunusual miRNA biogenesis pathway, which differs in pre-miRNA synthesis, nuclear-cytoplasmic transport, and guide strand selection, enables the development of shRNA expression constructs that produce a single 3p-siRNA.

Genome Regulation by Long Non-Coding RNAs

The discovery of extensive transcription of long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. Discussions will cover genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and both control and target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, long non-coding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. More recently, lncRNAs have been found to control post-transcriptional processes and signal transduction as well. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs in developmental patterning and cancer. Disclosures:Chang:RaNA Therapeutics: Membership on an entity's Board of Directors or advisory committees.

ADAR Regulates RNA Editing, Transcript Stability, and Gene Expression

SUMMARYAdenosine deaminases acting on RNA (ADARs) convert adenosine to inosine, which is then recognized as guanosine. To study the role of ADAR proteins in RNA editing and gene regulation, we sequenced and compared the DNA and RNA of human B cells. Then, we followed up the findings experimentally with siRNA knockdown and RNA and protein immunoprecipitations. The results uncovered over 60,000 A-to-G editing sites and several thousand genes whose expression levels are influenced by ADARs. Of these ADAR targets, 90% were identified. Our results also reveal that ADAR regulates transcript stability and gene expression through interaction with HuR (ELAVL1). These findings extend the role of ADAR and show that it cooperates with other RNA-processing proteins to regulate the sequence and expression of transcripts in human cells.

Three-state mechanism couples ligand and temperature sensing in riboswitches

Riboswitches are cis-acting gene-regulatory RNA elements that can function at the level of transcription, translation and RNA cleavage. The commonly accepted molecular mechanism for riboswitch function proposes a ligand-dependent conformational switch between two mutually exclusive states. According to this mechanism, ligand binding to an aptamer domain induces an allosteric conformational switch of an expression platform, leading to activation or repression of ligand-related gene expression. However, many riboswitch properties cannot be explained by a pure two-state mechanism. Here we show that the regulation mechanism of the adenine-sensing riboswitch, encoded by the add gene on chromosome II of the human Gram-negative pathogenic bacterium Vibrio vulnificus, is notably different from a two-state switch mechanism in that it involves three distinct stable conformations. We characterized the temperature and Mg(2+) dependence of the population ratios of the three conformations and the kinetics of their interconversion at nucleotide resolution. The observed temperature dependence of a pre-equilibrium involving two structurally distinct ligand-free conformations of the add riboswitch conferred efficient regulation over a physiologically relevant temperature range. Such robust switching is a key requirement for gene regulation in bacteria that have to adapt to environments with varying temperatures. The translational adenine-sensing riboswitch represents the first example, to our knowledge, of a temperature-compensated regulatory RNA element.

Deep parallel sequencing reveals conserved and novel miRNAs in gill and hepatopancreas of giant freshwater prawn

MicroRNAs (miRNAs) are ∼20–22 nucleotides, non protein-coding RNA regulatory genes that post-transcriptionally regulate many protein-coding genes, influencing critical biological and metabolic processes. While the number of known microRNA is increasing, there is currently no published data for miRNA from giant freshwater prawns, Macrobrachium rosenbergii (M. rosenbergii), a commercially cultured and economically important food species. In this study, we identified novel miRNAs in the gill and hepatopancreas of M. rosenbergii. Through a deep parallel sequencing analysis and an in silico data analysis approach, 327 miRNA families were identified from small RNA libraries with reference to both the de novo transcriptome of M. rosenbergii obtained from RNA-Seq and to miRBase (Release 18.0, November 2012). Based on the identified mature miRNA and recovered precursor sequences that form appropriate hairpin structures, three conserved miRNA (miR125, miR750, miR993) and 27 novel miRNA candidates encoding messenger-like non-coding RNA were identified. miR-125, miR-750, G-m0002/H-m0009, G-m0005, G-m0008/H-m0016, G-m0011/H-m0027 and G-m0015 were selected for experimental validation with stem-loop quantitative RT-PCR and were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.835178 for miRNA in gill, r = 0.724131 for miRNA in hepatopancreas). Using a combinatorial approach of pathway enrichment analysis and inverse expression relationship of miRNA and mRNA, four co-expressed novel miRNA candidates (G-m0005, G-m0008/H-m0016, G-m0011/H-m0027, and G-m0015) were found to be associated with energy metabolism. In addition, the expression of the three novel miRNA candidates (G-m0005, G-m0008/H-m0016, and G-m0011/H-m0027) were also found to be significantly reduced at 9 and 24 h post infection in M. rosenbergii challenged with infectious hypodermal and hematopoietic necrosis virus, suggesting a functional role of these miRNAs in crustacean immune defense.

Glucosamine and Glucosamine-6-phosphate Derivatives: Catalytic Cofactor Analogues for the glmS Ribozyme

Two analogues of glucosamine-6-phosphate (GlcN6P, 1) and five of glucosamine (GlcN, 2) were prepared for evaluation as catalytic cofactors of the glmS ribozyme, a bacterial gene-regulatory RNA that controls cell wall biosynthesis. Glucosamine and allosamine with 3-azido substitutions were prepared by SN2 reactions of the respective 1,2,4,6-protected sugars; final acidic hydrolysis afforded the fully deprotected compounds as their TFA salts. A 6-phospho-2-aminoglucolactam (31) was prepared from glucosamine in a 13-step synthesis, which included a late-stage POCl3-phosphorylation. A simple and widely applicable 2-step procedure with the triethylsilyl (TES) protecting group was developed to selectively expose the 6-OH group in N-protected glucosamine analogues, which provided another route to chemical phosphorylation. Mitsunobu chemistry afforded 6-cyano (35) and 6-azido (36) analogues of GlcN-(Cbz), and the selectivity for the 6-position was confirmed by NMR (COSY, HMBC, HMQC) experiments. Compound 36 was converted to the fully deprotected 6-azido-GlcN (37) and 2,6-diaminoglucose (38) analogues. A 2-hydroxylamino glucose (42) analogue was prepared via an oxaziridine (41). Enzymatic phosphorylation of 42 and chemical phosphorylation of its 6-OH precursor (43) were possible, but 42 and the 6-phospho product (44) were unstable under neutral or basic conditions. Chemical phosphorylation of the previously described 2-guanidinyl-glucose (46) afforded its 6-phospho analogue (49) after final deprotection.

Abstract SY31-03: Genome regulation by long noncoding RNAs.

Abstract The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serves as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs) provide an important new perspective on the centrality of RNA in gene regulation. An emerging theme from multiple model systems is that LncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators, and target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, long noncoding RNAs can function as modular scaffolds to specify higher order organization in RNP complexes and in chromatin states. I will discuss how this mode of regulation is particularly relevant to cancer and the maintenance of genome integrity. Citation Format: Howard Y. Chang. Genome regulation by long noncoding RNAs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY31-03. doi:10.1158/1538-7445.AM2013-SY31-03

Identification of PKM2 Interacting Proteins by Tandem Mass Spectrometry

Pyruvate Kinase M2 (PKM2) plays a key role in aerobic glycolysis and the metabolic phenotype that is critical for cancer cell growth and proliferation. Recent studies have demonstrated that PKM2 may have nonmetabolic functions like regulation of gene transcription and protein phosphorylation. We hypothesized that liquid chromatography tandem mass spectrometry (LC‐MS/MS) “interactome” analysis of PKM2 will identify additional functional roles of PKM2. We carried out immunoprecipitation (IP) of DEL cells stably expressing FLAG‐tagged wild type (WT) PKM2 and the Y105F mutated PKM2 (glycolytically active) and subjected the IP to LC‐MS/MS. A total of 274 proteins were detected in the WT PKM2 pull down. There were 57 proteins unique to WT PKM2 relative to the Y105F PKM2 mutant. We carried out validation via IP and western blot for three proteins involved in RNA metabolism and gene regulation (HMGA1, RNF20, and PTBP1). Interaction of these proteins was predominantly detected with the WT isoform and not the Y105F isoform. DAVID analysis showed that WT PKM2 interacted more with proteins involved in nuclear functions such as RNA metabolism and gene expression than its mutated counterpart. Our results indicate that PKM2 may have a role in RNA metabolism and gene regulation through its interaction with HMGA1, RNF20, and PTBP1.Funding support comes from the University of Michigan, Department of Pathology development funds.

Editorial Changes at the Journal of Molecular Evolution

On January 1, 2013 Martin Kreitman stepped down as editor-in-chief (EIC) of the Journal of Molecular Evolution after 15 years in this role. During this time the rich tradition of this journal has continued, and the readership base has broadened. These have been exciting times in the fields and sub-fields that connect to the topic of molecular evolution. The human genome was completely sequenced, and we have seen an explosion of genomic-wide data, the advent of high-throughput sequencing, fascinating developments in the interface between development and evolution (‘‘evo-devo’’), the burgeoning of an understanding of the role of small RNAs in gene regulation, rapid advances in high-throughput X-ray crystallization-based structure determination of both proteins and nucleic acids, sweeping advances in computational hardware and techniques, and the entrenchment of the Internet as the means by which data is stored, analyzed, and shared, and by which scientific publishing is accomplished. Through all of this, Marty has strove to bring the highest quality research to JME, but for personal reasons has chosen to step down as EIC and focus on other issues. I will become the new EIC for JME after having been an associate editor (AE) with the journal since 1996. I received my B.S. in Chemistry from the University of California, Berkeley in 1984 and stayed on to obtain a Master’s degree with Tom Jukes in the Space Sciences Laboratory at Berkeley in 1986, studying the evolution of the genetic code. I was Tom’s last graduate student and first became exposed to the rich field of molecular evolution—and to JME—during my stay in Tom’s lab. I then moved to UCLA to be one of the first Center for the Study of Evolution and the Origins of Life (CSEOL) Fellows at UCLA, a program founded by J. William Schopf. There I worked with Bob Wayne and Chuck Taylor on the molecular population genetics of canids and received my Ph.D. in 1990. From there I moved on to be a post-doctoral fellow at The Scripps Research Institute in La Jolla, CA, working with Jerry Joyce on the in vitro evolution of the Tetrahymena ribozyme. I then did a second post-doc with Mike Lynch at the University of Oregon studying phylogenetics and speciation in the freshwater microcrustacean Daphnia. After two tenure-track appointments at other universities, I arrived at Portland State University in Portland, Oregon in 2001 where I am now a full professor of Chemistry. My current research focuses on the use of catalytic RNAs to study self-replication, molecular cooperation, and the advent and evolution of genetic information during the origins of life on the Earth. Several Ph.D. students have graduated from my lab at Portland State, supported by funding by through various NASA programs such as Exobiology and Evolutionary Biology. This editorial change marks only the second one in JME’s history. The journal was founded in 1971, when Conrad Springer offered Emile Zuckerkandl, who along with Linus Pauling had originated the idea of the molecular clock, the position of EIC (Bernardi 2012). Zuckerkandl accepted, and with a staff of four editors and 41 editorial board members, the journal published its first issue in the summer of 1971. Among the four editors was Dick Dickerson, a pioneer in the study of DNA structure and coincidentally on my Ph.D. committee at UCLA. Another of the editors was Jack Lester King, co-author with Tom Jukes of a classic 1969 paper entitled ‘‘Non-Darwinian Evolution’’ (King and Jukes 1969), which helped to define the study of molecular evolution. The editorial board N. Lehman (&) Department of Chemistry, Portland State University, Portland, OR 97207, USA e-mail: jme1@pdx.edu; niles@pdx.edu

Structure and Functional Dynamics of Fluoride-Sensing Riboswitches

Riboswitches are gene-regulatory RNA motifs located in the 5' untranslated regions of certain bacterial mRNA's. Riboswitches regulate gene expression by binding a metabolite related to the downstream gene, causing a conformational change that alters the accessibility of the gene for either transcription or translation. An important class of riboswitches that bind fluoride (F-) has been identified recently in bacteria and archaea, shedding light on how these organisms regulate internal fluoride concentrations to mitigate toxicity. The crystal structure of the fluoride riboswitch from Thermophilus petrophila shows a binding pocket in which the F- ion is coordinated by three Mg2+ ions. However, how ligand recognition and RNA folding are coupled to selectively encapsulate F- is not fully understood. Here, single-molecule TIRF microscopy and FRET are used to gain insights into the functional dynamics of fluoride riboswitches. Individual fluorescently-labelled fluoride riboswitches are immobilized on a quartz microscope slide, and the change in FRET efficiency between the fluorophors is used to study the ligand-binding mechanism and other cation- or denaturant-dependent structural transitions.

Introduction: The Use of Animals Models to Advance Epigenetic Science

Introduction: The Use of Animals Models to Advance Epigenetic Science

Future Prospects for Pharmacogenetics in the Quest for Personalized Medicine

Understanding pharmacogenetic differences in drug response and tolerability has been an important area of research in personalized medicine, but the clinical utility of pharmacogenetics testing has not been established. Identification of genetic polymorphisms due to single nucleotide polymorphisms is the most common approach, but this does not take into account the potential relevance of copy number variants, noncoding RNA gene regulation, gene-gene and gene-interactions, and epigenetic modifications, which increase the complexity of pharmacogenomics research.

Genome Regulation by Long Noncoding RNAs

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. The discovery of extensive transcription of large RNA transcripts that do not code for proteins, termed long noncoding RNAs (lncRNAs), provides an important new perspective on the centrality of RNA in gene regulation. Here, we discuss genome-scale strategies to discover and characterize lncRNAs. An emerging theme from multiple model systems is that lncRNAs form extensive networks of ribonucleoprotein (RNP) complexes with numerous chromatin regulators and then target these enzymatic activities to appropriate locations in the genome. Consistent with this notion, lncRNAs can function as modular scaffolds to specify higher-order organization in RNP complexes and in chromatin states. The importance of these modes of regulation is underscored by the newly recognized roles of long RNAs for proper gene control across all kingdoms of life.

A Second-Generation MicroRNA-Based Assay for Diagnosing Tumor Tissue Origin

Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Sequencing and analysis of four BAC clones containing innate immune genes from the Zhikong scallop (Chlamys farreri)

The sequencing of BAC clones (~100kb) can reveal some characteristics of a genome that are challenging to obtain based on short sequences. Additionally, although the immune genes of the Zhikong scallop (Chlamys farreri) have been studied widely, few analyses have been conducted at the DNA level. In this study, four C. farreri BAC clones containing innate immune genes, including hsp70, lgbp (lipopolysaccharide and beta-1,3-glucan binding protein), serine protease and a gene with an immunoglobulin-like domain, were sequenced and analyzed both to explore the genomic characteristics of C. farreri based on long DNA sequences and to promote the study of C. farreri immune genes at the DNA level. The total length of the four BACs was 389.98kb. A total of 34 genes were predicted in these sequences, and several features of protein-coding regions in the C. farreri genome were inferred based on this information. Two LGBP genes were located close together in a 22-kb region in one BAC clone, indicating the physical linkage of some immune genes in C. farreri. A cluster of membrane transport genes was also observed; these genes might play important roles in eliminating toxins in C. farreri, which lives as a filter feeder. Further analysis showed 15.43% of the BAC sequence was repetitive. Tandem repeats were the most abundant repeat type, followed by transposable elements. A total of 31 SSRs were predicted in the four BACs. An IS10 family transposon was identified, and a suspected regulatory non-coding RNA gene for this transposon (RNA-OUT) was observed to overlap with it complementarily. This work will promote future studies on the genomics, immune system and non-coding regions of C. farreri.