Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation.

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP‐based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell‐free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

Funding Information: Acknowledgements This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Green Technology Project EF-1004). We are grateful to Dr. Takuji Sasaki for his encouragement throughout the project and his excellent advice on the improvement of this manuscript, and to Dr. Shoshi Kikuchi for providing useful information on rice FL-cDNAs. We thank Professors Kokichi Hinata, Atsushi Hirai, Hiroshi Kamada and Masashi Ugaki for their encouragement, critical comments and helpful suggestions, and Drs. Hisato Okuizumi and Hiroyuki Kawahigashi for their administrative support throughout the project. We also thank Mayumi Akagawa, Hiroko Abe, Keiko Mori, Etsuko Sugai, Yumiko Nakane, Ken-ichi Watanabe, Mayumi Takeya, and Kana Miyata for their technical assistance; the members of the Technical Support Section of the National Institute of Agrobiological Sciences for their help in the care of the FOX-rice plants; Haruko Onodera and Kazuko Ono for their technical assistance and advice on rice transformation; Inplanta Innovations Inc. for their technical help on the construction of theThe latest report has estimated the number of rice genes to be ∼32 000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13 980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12 000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene, confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.publishersversionPeer reviewe

Site-specific isotope labeling of long RNA for structural and mechanistic studies

A site-specific isotope labeling technique of long RNA molecules was established. This technique is comprised of two simple enzymatic reactions, namely a guanosine transfer reaction of group I self-splicing introns and a ligation with T4 DNA ligase. The trans-acting group I self-splicing intron with its external cofactor, ‘isotopically labeled guanosine 5′-monophosphate’ (5′-GMP), steadily gave a 5′-residue-labeled RNA fragment. This key reaction, in combination with a ligation of 5′-remainder non-labeled sequence, allowed us to prepare a site-specifically labeled RNA molecule in a high yield, and its production was confirmed with 15N NMR spectroscopy. Such a site-specifically labeled RNA molecule can be used to detect a molecular interaction and to probe chemical features of catalytically/structurally important residues with NMR spectroscopy and possibly Raman spectroscopy and mass spectrometry

Mass spectrometric detection of differential methylation of hnRNP A2/B1 paralogs in neuronal cells and cell lines

Background The heterogeneous nuclear ribonucleoprotein paralogs, A1, A2/B1 and A3 (hnRNPs A/B) share a high degree of sequence similarity and may have closely related roles in processes such as RNA packaging, alternative splicing, telomere maintenance and cytoplasmic trafficking. hnRNPs A/B exhibit different patterns of sub-nuclear localization, suggesting that these paralogs have diverged to perform functionally distinct roles. Additional functional diversity is created by posttranslational modifications, coupled with the generation of alternatively spliced isoforms. We have investigated hnRNP A2 post-translational modifications in native rat brain, rat B104, human HeLa and human SH-SY5Y transformed cells. Methods We have used a combination of ESI-QTOF and MALDI-TOF/TOF mass spectrometry, as well as Edman degradation to identify and confirm methylation patterns of hnRNP A2/B1. Results Our results using both mass spectrometry and Edman degradation clearly show that hnRNP A2 is NG,NG-dimethylated on a single arginine residue, Arg254, encoded within the alternatively spliced exon 9. This is in contrast to hnRNP A1, which has four of its five RGG motifs NG,NG-dimethylated on the arginine residues. In native rat brain, Arg254 is almost completely dimethylated, while in rat and human cell lines, a greater proportion of - 108 - this arginine residue is either unmethylated or monomethylated. Conclusions This conserved modification of Arg254 in hnRNP A2 suggests that hnRNP A2 is functionally regulated by dimethylation in a manner that distinguishes it from hnRNP A1. We are currently expanding our analysis of hnRNP proteins to include zebrafish to determine the conservation of methylation status across organisms

Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation

Abstract RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA–RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future

A novel NAC transcription factor, IDEF2, which recognizes the iron deficiency-responsive element 2, regulates genes involved in iron homeostasis

Iron (Fe) is essential for most living organisms, and thus Fe deficiency poses a major abiotic stress in crop production. Plants induce Fe utilization systems under conditions of low-Fe availability, but the molecular mechanisms of gene regulation under Fe deficiency remain largely unknown. We identified a novel transcription factor in rice and barley, IDEF2, which specifically binds to the Fe deficiency-responsive cis-acting element 2 (IDE2), using yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets (CASTing) experiment revealed that IDEF2 predominantly recognizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the core binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by RNA interference (RNAi) and chimeric repressor gene-silencing technology (CRES-T) caused aberrant Fe distribution between shoots and roots in rice grown hydroponically under Fe-sufficient or deficient conditions. These results reveal novel cis-element/trans-factor interactions that are functionally associated with iron homeostasis