Importance of experimental information (metadata) for archived sequence data: case of specific gene bias due to lag time between sample harvest and RNA protection in RNA sequencing

Large volumes of high-throughput sequencing data have been submitted to the Sequencing Read Archive (SRA). The lack of experimental metadata associated with the data makes reuse and understanding data quality very difficult. In the case of RNA sequencing (RNA-Seq), which reveals the presence and quantity of RNA in a biological sample at any moment, it is necessary to consider that gene expression responds over a short time interval (several seconds to a few minutes) in many organisms. Therefore, to isolate RNA that accurately reflects the transcriptome at the point of harvest, raw biological samples should be processed by freezing in liquid nitrogen, immersing in RNA stabilization reagent or lysing and homogenizing in RNA lysis buffer containing guanidine thiocyanate as soon as possible. As the number of samples handled simultaneously increases, the time until the RNA is protected can increase. Here, to evaluate the effect of different lag times in RNA protection on RNA-Seq data, we harvested CHO-S cells after 3, 5, 6, and 7 days of cultivation, added RNA lysis buffer in a time course of 15, 30, 45, and 60 min after harvest, and conducted RNA-Seq. These RNA samples showed high RNA integrity number (RIN) values indicating non-degraded RNA, and sequence data from libraries prepared with these RNA samples was of high quality according to FastQC. We observed that, at the same cultivation day, global trends of gene expression were similar across the time course of addition of RNA lysis buffer; however, the expression of some genes was significantly different between the time-course samples of the same cultivation day; most of these differentially expressed genes were related to apoptosis. We conclude that the time lag between sample harvest and RNA protection influences gene expression of specific genes. It is, therefore, necessary to know not only RIN values of RNA and the quality of the sequence data but also how the experiment was performed when acquiring RNA-Seq data from the database.

Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5′ ends corresponding to transcription start sites (TSS), as well as 1628 processed 5′ ends and 1299 3′ ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5′ and 3′ ends, contrasting with the prevailing description of discrete 5′ termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1–1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1–1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.

Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

ABSTRACTChloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-Seq. UsingArabidopsis thalianaas a model, we catalogued >215 primary 5’ ends corresponding to transcription start sites (TSS), as well as 1,628 processed 5’ ends and 1,299 3’ ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5’ and 3’ ends, contrasting with the prevailing description of discrete 5’ termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-Seq was also implemented forpnp1-1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2,000 termini were altered inpnp1-1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-Seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.

Ty3 Nucleocapsid Controls Localization of Particle Assembly

ABSTRACT Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX2CX4HX4C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.

Ty3 Capsid Mutations Reveal Early and Late Functions of the Amino-Terminal Domain

ABSTRACT The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.