Transfection of Small Noncoding RNAs into Arabidopsis thaliana Protoplasts.

Polyethylene glycol transfection of plant protoplasts represents an efficient method to incorporate foreign DNA and study transient gene expression. Here, we describe an optimized protocol to deliver small noncoding RNAs into Arabidopsis thaliana protoplasts. An example of application is provided by demonstrating the incorporation of a 20 nt long small noncoding RNA deriving from the 5' extremity of an A. thaliana cytosolic alanine tRNA into freshly isolated protoplasts.

Arabidopsis tRNA-derived fragments as potential modulators of translation.

Transfer RNA-derived fragments (tRFs) exist in all branches of life. They are involved in RNA degradation, regulation of gene expression, ribosome biogenesis. In archaebacteria, kinetoplastid, yeast, and human cells, they were also shown to regulate translation. In Arabidopsis, the tRFs population fluctuates under developmental or environmental conditions but their functions are yet poorly understood. Here, we show that populations of long (30-35 nt) or short (19-25 nt) tRFs produced from Arabidopsis tRNAs can inhibit in vitro translation of a reporter gene. Analysing a series of oligoribonucleotides mimicking natural tRFs, we demonstrate that only a limited set of tRFs possess the ability to affect protein synthesis. Out of a dozen of tRFs, only two deriving from tRNAAla(AGC) and tRNAAsn(GUU) strongly attenuate translation in vitro. Contrary to human tRF(Ala), the 4 Gs present at the 5' extremity of Arabidopsis tRF(Ala) are not implicated in this inhibition while the G18 and G19 residues are essential. Protein synthesis inhibition by tRFs does not require complementarity with the translated mRNA but, having the capability to be associated with polyribosomes, tRFs likely act as general modulation factors of the translation process in plants.

A case report: mechanical mitral valve thrombosis in pregnancy.

BACKGROUND: Pregnancy in women with mechanical valves has a high risk of both valve thrombosis and bleeding as well as adverse effects on the foetus. There is limited data on achieving optimal anticoagulation in pregnancy and management of valve thrombosis, to achieve a successful foetal outcome, while prioritizing the mother's health. While warfarin may carry a lower risk of valve thrombosis, warfarin is teratogenic in the first trimester and is associated with increased foetal loss throughout the pregnancy. Heparin does not cross the placenta but is associated with increased maternal morbidity and mortality. CASE SUMMARY: We describe the case of a pregnant patient with thrombosis of a mechanical mitral valve presenting with an embolic stroke at 22 weeks of pregnancy. The stroke was treated with clot retrieval and resulted in no residual neurological deficit. Two previous pregnancies had been managed with low molecular weight heparin, and both resulted in foetal loss. The patient was determined to continue this pregnancy. She was treated with intravenous unfractionated heparin during the remainder of the pregnancy. She developed worsening heart failure due to persisting valve thrombosis despite maintenance of therapeutic anticoagulation. The patient deteriorated rapidly prior to a planned early elective delivery. Emergency Caesarean section was required followed by valve replacement using extracorporeal membrane oxygenation support with an ultimately successful maternal and foetal outcome. Anticoagulation regimes and treatment of mechanical valve thrombosis in pregnancy are discussed. DISCUSSION: The management of pregnant patients with mechanical valves is complex, especially when valve thrombosis and other complications occur. A multidisciplinary approach is essential and in this case led to successful outcome.

Plant RNases T2, but not Dicer-like proteins, are major players of tRNA-derived fragments biogenesis.

RNA fragments deriving from tRNAs (tRFs) exist in all branches of life and the repertoire of their biological functions regularly increases. Paradoxically, their biogenesis remains unclear. The human RNase A, Angiogenin, and the yeast RNase T2, Rny1p, generate long tRFs after cleavage in the anticodon region. The production of short tRFs after cleavage in the D or T regions is still enigmatic. Here, we show that the Arabidopsis Dicer-like proteins, DCL1-4, do not play a major role in the production of tRFs. Rather, we demonstrate that the Arabidopsis RNases T2, called RNS, are key players of both long and short tRFs biogenesis. Arabidopsis RNS show specific expression profiles. In particular, RNS1 and RNS3 are mainly found in the outer tissues of senescing seeds where they are the main endoribonucleases responsible of tRNA cleavage activity for tRFs production. In plants grown under phosphate starvation conditions, the induction of RNS1 is correlated with the accumulation of specific tRFs. Beyond plants, we also provide evidence that short tRFs can be produced by the yeast Rny1p and that, in vitro, human RNase T2 is also able to generate long and short tRFs. Our data suggest an evolutionary conserved feature of these enzymes in eukaryotes.

The nuclear and organellar tRNA-derived RNA fragment population in Arabidopsis thaliana is highly dynamic.

In the expanding repertoire of small noncoding RNAs (ncRNAs), tRNA-derived RNA fragments (tRFs) have been identified in all domains of life. Their existence in plants has been already proven but no detailed analysis has been performed. Here, short tRFs of 19-26 nucleotides were retrieved from Arabidopsis thaliana small RNA libraries obtained from various tissues, plants submitted to abiotic stress or fractions immunoprecipitated with ARGONAUTE 1 (AGO1). Large differences in the tRF populations of each extract were observed. Depending on the tRNA, either tRF-5D (due to a cleavage in the D region) or tRF-3T (via a cleavage in the T region) were found and hot spots of tRNA cleavages have been identified. Interestingly, up to 25% of the tRFs originate from plastid tRNAs and we provide evidence that mitochondrial tRNAs can also be a source of tRFs. Very specific tRF-5D deriving not only from nucleus-encoded but also from plastid-encoded tRNAs are strongly enriched in AGO1 immunoprecipitates. We demonstrate that the organellar tRFs are not found within chloroplasts or mitochondria but rather accumulate outside the organelles. These observations suggest that some organellar tRFs could play regulatory functions within the plant cell and may be part of a signaling pathway.

Surveillance and cleavage of eukaryotic tRNAs.

Beyond their central role in protein synthesis, transfer RNAs (tRNAs) have many other crucial functions. This includes various roles in the regulation of gene expression, stress responses, metabolic processes and priming reverse transcription. In the RNA world, tRNAs are, with ribosomal RNAs, among the most stable molecules. Nevertheless, they are not eternal. As key elements of cell function, tRNAs need to be continuously quality-controlled. Two tRNA surveillance pathways have been identified. They act on hypo-modified or mis-processed pre-tRNAs and on mature tRNAs lacking modifications. A short overview of these two pathways will be presented here. Furthermore, while the exoribonucleases acting in these pathways ultimately lead to complete tRNA degradation, numerous tRNA-derived fragments (tRFs) are present within a cell. These cleavage products of tRNAs now potentially emerge as a new class of small non-coding RNAs (sncRNAs) and are suspected to have important regulatory functions. The tRFs are evolutionarily widespread and created by cleavage at different positions by various endonucleases. Here, we review our present knowledge on the biogenesis and function of tRFs in various organisms.

Structural deterioration of the cryopreserved mitral homograft valve.

OBJECTIVE: The aim of this study was to evaluate the long-term fate of the cryopreserved mitral homograft focusing on structural valve deterioration. METHODS: Homograft replacement of the mitral valve was performed in 106 patients. The causes of mitral disease were rheumatic disease (n=75), endocarditis (n=24), and others (n=7). There were 40 partial homografts and 66 total homografts. RESULTS: Mean follow-up was 9.3+4.7 years (up to 17.8 years). There were 5 early (<3 months) and 15 late deaths. There have been 5 early (<3 months) and 30 late reoperations. Five patients had endocarditis, and 5 patients had an ischemic/hemorrhagic event. Compared with baseline, follow-up echography showed progression of mitral regurgitation grade (from 0.4 to 1.3; P<.001) with stenosis (elevated gradient: from 3.9 to 7.0 mm Hg; P<.001) and decreased valve area (from 2.3 to 1.7 cm2, P<.001). Freedom from structural valve deterioration was 90%, 76%, and 65% at 5, 10, and 15 years, respectively. Structural valve deterioration was more frequent in total homografts (P=.018 vs partial homografts) and in case of pregnancy (P=.016 vs no pregnancy). Stenosis related to structural valve deterioration was more pronounced for age less than 40 years (P=.03) and ring size 30 mm or less (P=.002). Pathologic analysis of the explanted homografts almost invariably showed dense fibrosis with calcification and no cellularity. CONCLUSIONS: Mitral homografting was accomplished with early echographic results similar to those of valve repair. Structural valve deterioration produced mixed stenosis with insufficiency, and its incidence was comparable to that of bioprostheses structural valve deterioration. An improvement in the preservation mode of valvular homografts is warranted.