In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

RNA editing of the col mRNA throughout the life cycle of Physarum polycephalum

Editing of RNA via the insertion, deletion or substitution of genetic information affects gene expression in a variety of systems. Previous characterization of the Physarum polycephalum cytochrome c oxidase subunit I (col) mRNA revealed that both nucleotide insertions and base substitutions occur during the maturation of this mitochondrial message. Both types of editing are known to be developmentally regulated in other systems, including mammals and trypanosomatids. Here we show that the col mRNA present in Physarum mitochondria is edited via specific nucleotide insertions and C to U conversions at every stage of the life cycle. Primer extension sequencing of the RNA indicates that this editing is both accurate and efficient. Using a sensitive RT-PCR assay to monitor the extent of editing at individual sites of C insertion, we estimate that greater than 98% of the steady-state amount of col mRNA is edited throughout the Physarum developmental cycle.     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract

The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.     

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.     

Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.     

Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.     

单碱基编辑系统介导欧拉藏绵羊GDF9、FecB多胎基因的定点突变

本研究旨在利用单碱基编辑系统(single base editing system)实现欧拉藏绵羊成纤维细胞FecB和GDF9基因靶位点A到G和C到T的碱基替换并检测其编辑效率。首先设计合成靶向欧拉藏绵羊FecB和GDF9基因的sgRNA序列,再分别连接至epi-ABEmax、epi-BE4max质粒,构建载体并电转至欧拉藏绵羊成纤维细胞,最后对阳性细胞FecB和GDF9基因进行Sanger测序鉴定靶位点突变结果,并通过T-A克隆估算单碱基编辑系统的编辑效率。结果显示获得了靶向欧拉藏绵羊FecB和GDF9基因的sgRNA,并构建使欧拉藏绵羊FecB和GDF9基因单碱基突变的载体,FecB基因靶位点编辑效率为39.13%,GDF9基因靶位点(G260、G721、G1184)编辑效率分别为10.52%、26.67%和8.00%。本研究运用单碱基编辑系统在欧拉藏绵羊成纤维细胞上实现了FecB和GDF9基因靶位点突变,为改良欧拉藏绵羊一胎多羔的繁殖性状奠定理论基础。     

RNA干扰技术在果蝇中的应用

RNA干扰是双链RNA特异诱导的转录后期基因沉默.该技术随着不断完善而越来越被广泛地运用于果蝇的功能基因组研究上,双链RNA已经成为果蝇中功能基因的一个十分有效的抑制子,势必使RNA干扰技术成为研究果蝇体内基因功能的强有力的反向遗传学研究技术.