Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler.

BACKGROUND: The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage. RESULTS: We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli. CONCLUSION: The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

Single-Cell Transcriptome Analysis of Olfactory Sensory Neurons

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements

Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.

In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.