SnapShot: RNA Structure Probing Technologies.

Chemical probing coupled to high-throughput sequencing offers a flexible approach to uncover many aspects of RNA structure relevant to its cellular function. With a wide variety of chemical probes available that each report on different features of RNA molecules, a broad toolkit exists for investigating in vivo and in vitro RNA structure and interactions with other molecules.

Dynamic RNA Modifications in Gene Expression Regulation.

Over 100 types of chemical modifications have been identified in cellular RNAs. While the 5' cap modification and the poly(A) tail of eukaryotic mRNA play key roles in regulation, internal modifications are gaining attention for their roles in mRNA metabolism. The most abundant internal mRNA modification is N6-methyladenosine (m6A), and identification of proteins that install, recognize, and remove this and other marks have revealed roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes. Abundant noncoding RNAs such as tRNAs, rRNAs, and spliceosomal RNAs are also heavily modified and depend on the modifications for their biogenesis and function. Our understanding of the biological contributions of these different chemical modifications is beginning to take shape, but it's clear that in both coding and noncoding RNAs, dynamic modifications represent a new layer of control of genetic information.

Determination of tRNA aminoacylation levels by high-throughput sequencing.

Transfer RNA (tRNA) decodes mRNA codons when aminoacylated (charged) with an amino acid at its 3' end. Charged tRNAs turn over rapidly in cells, and variations in charged tRNA fractions are known to be a useful parameter in cellular responses to stress. tRNA charging fractions can be measured for individual tRNA species using acid denaturing gels, or comparatively at the genome level using microarrays. These hybridization-based approaches cannot be used for high resolution analysis of mammalian tRNAs due to their large sequence diversity. Here we develop a high-throughput sequencing method that enables accurate determination of charged tRNA fractions at single-base resolution (Charged DM-tRNA-seq). Our method takes advantage of the recently developed DM-tRNA-seq method, but includes additional chemical steps that specifically remove the 3'A residue in uncharged tRNA. Charging fraction is obtained by counting the fraction of A-ending reads versus A+C-ending reads for each tRNA species in the same sequencing reaction. In HEK293T cells, most cytosolic tRNAs are charged at >80% levels, whereas tRNASer and tRNAThr are charged at lower levels. These low charging levels were validated using acid denaturing gels. Our method should be widely applicable for investigations of tRNA charging as a parameter in biological regulation.

tRNA base methylation identification and quantification via high-throughput sequencing.

Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative "Modification Index" (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore.

Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

TRIM5α variations influence transduction efficiency with lentiviral vectors in both human and rhesus CD34(+) cells in vitro and in vivo.

Human immunodeficiency virus type 1 (HIV-1) vectors can transduce human hematopoietic stem cells (HSC), but transduction efficiency varies among individuals. The innate immune factor tripartite motif-containing protein 5α (TRIM5α) plays an important role for restriction of retroviral infection. In this study, we examined whether TRIM5α could account for variations in transduction efficiency using both an established rhesus gene therapy model and human CD34(+) cell culture. Evaluation of TRIM5α genotypes (Mamu-1, -2, -3, -4, -5, and TrimCyp) in 16 rhesus macaques that were transplanted with transduced CD34(+) cells showed a significant correlation between TRIM5α Mamu-4 and high gene marking in both lymphocytes and granulocytes 6 months after transplantation. Since significant human TRIM5α coding polymorphisms were not known, we evaluated TRIM5α expression levels in human CD34(+) cells from 14 donors. Three days after HIV-1 vector transduction, measured transduction efficiency varied significantly among donors and was negatively correlated with TRIM5α expression levels. In summary, transduction efficiency in both rhesus and human CD34(+) cells was influenced by TRIM5α variations (genotypes and expression levels). Our findings are important for both understanding and mitigating the variability of transduction efficiency for rhesus and human CD34(+) cells.

High-efficiency transduction of rhesus hematopoietic repopulating cells by a modified HIV1-based lentiviral vector.

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

RNA modification landscape of the human mitochondrial tRNALys regulates protein synthesis.

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N1-methyladenosine (m1A) modification is missing at position 58 in the mitochondrial tRNALys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNALys, we demonstrated the importance of the m1A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.

Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number.

Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.Molecular Therapy-Nucleic Acids (2013) 2, e122; doi:10.1038/mtna.2013.49; published online 17 September 2013.