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PubMed1997

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.

Animals↗
PubMed2025

Genome editing research initiatives and regulatory landscape of genome edited crops in India.

Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.

Crops, Agricultural↗
PubMed2025

Preparation of high-purity RNPs of CRISPR-based DNA base editors.

Since their introduction, CRISPR-based DNA base editors (BEs) have become essential in the field of precision genome editing, revolutionizing the correction of pathogenic SNPs for both basic research and therapeutic applications. As this technology advances, more laboratories are implementing these tools into their workflow. The delivery of BEs as BE-guide RNA complexes (RNPs), rather than as mRNA or plasmids, has been shown to exhibit lower off-target effects, establishing it as the preferred method of delivery. However, there are no protocols describing in detail how to obtain high-purity and highly active BE RNPs. Here, we offer a comprehensive guide for the expression, purification, RNP reconstitution, and in vitro activity assessment of TadA-based BEs. The protocol includes guidance on performing activity assays using commercial denaturing gels, which is convenient and uses standard molecular biology equipment. This allows for rapid quality control testing of reconstituted BE RNPs prior to more expensive and time-consuming in vivo genome editing experiments. Overall, this protocol aims to empower more laboratories to generate tailored BE RNPs for diverse in vitro and in vivo applications.

Gene Editing↗
PubMed2001

RNA editing-associated protein 1 is an RNA binding protein with specificity for preedited mRNA.

RNA editing in the mitochondria of kinetoplastids involves the addition and deletion of uridines at specific sites as directed by guide RNAs (gRNAs). Ample evidence shows that ribonucleoprotein (RNP) complexes carry out this posttranscriptional processing. One component of RNA editing complexes is REAP-1, a protein of previously unknown function found primarily in mRNA containing editing complexes. We now show that REAP-1 is an RNA binding protein and map the binding activity to the amino-terminal third of the protein. REAP-1 binds to poly(G) and single-stranded guanosine rich RNAs. Data presented here demonstrates that preedited RNAs are the preferred substrate for REAP-1. The results suggest a model in which the role of REAP-1 is to bring preedited mRNAs into the editing complex.

Animals↗
PubMed2006

Prospective, randomized, open-label, fixed-dose, crossover study to establish preference of patients with erectile dysfunction after taking the three PDE-5 inhibitors.

INTRODUCTION: We conducted a prospective, randomized, open-label, fixed-dose preference study, with a crossover design, using sildenafil, vardenafil, and tadalafil. AIM: To assess patient preference for sildenafil (100 mg), vardenafil (20 mg), and tadalafil (20 mg) for the treatment of erectile dysfunction. Secondary objectives included finding out whether patients would follow treatment with a second or third option, in the event that the preferred drug was not available, and to assess side effects. MAIN OUTCOME MEASURES: Patient preference for any treatment, and evaluation of the elements that patients would assess when choosing one of these drugs. MATERIAL AND METHODS: Sildenafil (100 mg), vardenafil (20 mg), and tadalafil (20 mg) were taken at least six times over a period of 45-60 days with a washout period of 7 days. A total of 132 patients were enrolled to achieve a valid sample of 90 cases (15 per randomized group, total of six groups). Enrolled patients had mild to moderate erectile function. RESULTS: The International Index of Erectile Function (IIEF) score improved from baseline and was statistically significant in all cases (P < 0.0001). When we compared the IIEF scores, we found a statistically significant difference between tadalafil and vardenafil (P = 0.0002) favoring the former; similar results were obtained with the Erectile Dysfunction Inventory for Treatment Satisfaction (EDITS) Questionnaire (P = 0.000075). We also found a significant difference (P = 0.012) between tadalafil and sildenafil, again in favor of the former. In assessing drug preference, 25 patients (27.77%) chose sildenafil, 18 (20%) vardenafil, and 47 (52.22%) tadalafil. A total of 94% of patients would be willing to take another drug if the preferred choice was not available. All drugs were well tolerated. CONCLUSIONS: Although this is a preference study based on subjective elements, statistically significant differences comparing the IIEF score and the EDITS Questionnaire lead us to believe that beyond patients' subjective preference per se, said preference is probably also based on a genuinely superior response to one drug over another.

Aged↗
PubMed1997

A heterologous maize rpoB editing site is recognized by transgenic tobacco chloroplasts.

Single nucleotides in plant chloroplast transcripts are edited from the genomically encoded C to U, often resulting in changes of the encoded protein sequence. Site-specific trans-acting factors are postulated to direct the selection of edited residues. In order to further define cis sequences required for RNA editing, we investigated whether two editing sites present in maize rpoB mRNA would be recognized by the editing machinery of transformed tobacco chloroplasts. A 93-nucleotide (nt) segment surrounding site I is sufficient to direct editing of the maize sequence in tobacco chloroplasts. However, an 86-nt segment surrounding maize site IV (which is genomically encoded as a T in tobacco) does not confer editing of this site, suggesting that trans-acting factors necessary for recognition of site IV are not present in tobacco. The maize sequences surrounding site I were found to compete with the endogenous rpoB for a depletable trans factor and to reduce editing of endogenous site I. The presence of exogenous maize site I was also found to decrease editing of endogenous tobacco site II, indicating that there is a shared aspect of editing for some closely spaced editing sites.

Base Sequence↗
PubMed2006

Maintenance of plastid RNA editing activities independently of their target sites.

RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell.

Base Sequence↗
PubMed1995

Rapid corepressor exchange from the trp-repressor/operator complex: an NMR study of [ul-13C/15N]-L-tryptophan.

[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.

Bacterial Proteins↗
PubMed2006

Diagnostic, treatment, and demographic factors influencing survival in a population-based study of adult glioma patients in the San Francisco Bay Area.

We compare survival estimates for population-based glioma cases by using two diagnostic coding schemes, (1) the International Classification of Diseases, Oncology, second edition (ICD-O-2) as reported by the Surveillance, Epidemiology, and End Results (SEER) program and (2) central neuropathology review diagnosis based on the World Health Organization II classification. In addition, among review categories, we estimate survival in relation to several patient demographic and treatment factors. Eligible cases included adults residing in the San Francisco Bay SEER Area with newly diagnosed, histologically confirmed glioma during the years 1991-1994 and 1997-1999. The study group included participating subjects for whom subsequent central neuropathology review confirmed glioma. We determined treatments, vital status, and other factors by using registry, interview, medical record, and active follow-up data. Survival differences between anaplastic astrocytoma (AA) and astrocytoma were apparent from review diagnoses (median months of survival for AA, 13.0 [95% CI, 9.9-19.5], and astrocytoma, 101.3 [95% CI lower limit, 42.1; upper limit not yet reached]), but not with ICD-O-2 diagnoses reported by SEER (median months of survival for AA, 16.6 [95% CI, 12.0-20.7], and astrocytoma, not otherwise specified, 17.2 [95% CI, 10.6-71.6]). This finding emphasizes the need for improvements in coding for nonglioblastoma astrocytomas to provide better population survival estimates. When review diagnosis was used, younger age and resection (vs. biopsy) were statistically significant for all histology groups analyzed by multivariable Cox proportional hazard models. Additional statistically significant variables were as follows: among 517 glioblastoma patients, radiation treatment and being married; among 105 AA patients, inclusion of chemotherapy in the initial treatment; and among 106 patients with nonanaplastic oligodendroglial tumors, college education. Further consideration of impact of marital status, education, and other social factors in glioma survival may be warranted.

Adult↗
PubMed1995

RNA editing in wheat mitochondria proceeds by a deamination mechanism.

Most if not all mitochondrial messenger RNAs from seed plants undergo a post-transcriptional modification (RNA editing) involving the conversion of some cytidine residues to uridine. Using a molecular hybridization approach, an in vitro RNA editing system, able to faithfully reproduce the in vivo observed C to U changes of subunit 9 (atp9) of wheat mitochondrial ATP synthase mRNA, has been described [Araya et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1040-1044]. In this work we extend these studies to better understand the biochemical mechanism of this process. RNA editing was analysed by P1 nuclease digestion of the reaction product followed by thin layer chromatography. Experiments performed with unedited [3H]RNA labelled on the base and with unedited [32P]RNA labelled at the alpha-phosphate of cytidine residues, indicate that plant mitochondrial RNA editing operates through a deamination mechanism.

Base Sequence↗
PubMed2003

The central tolerance response to male antigen in normal mice is deletion and not receptor editing.

It is widely accepted that developing T cells can undergo clonal deletion in the thymus in response to a high affinity self-Ag. This is largely based on studies of TCR transgenics. However, encounter with high affinity self-Ag can also result in receptor editing in TCR transgenic models. Because all TCR transgenics display ectopic receptor expression, the tolerance mechanism that predominates in normal mice remains an open question. When self-Ag drives receptor editing during T cell development, one expects to find in-frame, self-reactive TCRalpha joins on TCR excision circles (TRECs), which are the products of secondary V/J recombination in the TCRalpha locus. Such joins are not expected if clonal deletion occurs, because the progenitor cell would be eliminated by apoptosis. To test the relative utilization of receptor editing vs clonal deletion, we determined the frequency of in-frame, male-specific joins on TRECs in male and female HYbeta transgenic mice. In comparison with female HYbeta transgenic mice, our analysis showed a lower frequency of TRECs with male-reactive V17J57 joins in male mice. Thus, it would appear that receptor editing is not a predominant tolerance mechanism for this self-Ag.

Animals↗
PubMed2006

Mono- and dicotyledonous plant-specific RNA editing sites are correctly edited in both in organello systems.

We set out to analyse the phylogenetic distribution of cox2 RNA editing sites. Database searches have revealed the presence of mono- and dicotyledonous-specific RNA editing sites. Therefore, to better understand tRNA editing system in plants, we developed a new dicotyledonous in organello RNA editing system using cauliflower mitochondria and analysed the transcription of the cox2 gene for both maize and Arabidopsis. These results were compared with those obtained from a maize mitochondrial in organello system. Surprisingly, both the mono- and dicotyledonous cox2 transcripts were efficiently edited in the mitochondrial cauliflower and maize in organello systems, respectively, even for RNA editing sites not present in the endogenous cox2 sequences. Taken together, our observations support a self-guiding-transcript model for RNA editing in higher plants.

Arabidopsis↗
PubMed1994

A new type of RNA editing. 5S ribosomal DNA transcripts are edited to mature 5S rRNA.

The primary structure of the 5S rRNA from Arabidopsis thaliana was determined. A comparison of this nucleotide sequence with that of 5S rRNA gene showed two differences. Furthermore, we compared all plant 5S rRNA and 5S rDNA sequences known to date and found that lack of colinearity is widespread among higher plant 5S rRNAs and occurred mainly in the double stranded regions. To explain this, we suggest a mechanism which converts putative products of pseudogene ("cryptogene") to mature 5S rRNA molecule. This kind of editing mechanism functions primarily in stems of 5S rRNA in order, to correct mispairing and thereby restoring the Watson-Crick base pairs. This idea could explain why so many different 5S rRNA genes or gene like sequences and only one 5S rRNA species exist in the plant cell. The editing can serve as a new mechanism of regulation of 5S rRNA synthesis in addition to transcription of 5S rRNA gene.

Base Sequence↗