Arabidopsis thaliana cbp80, c2h2, and flk Knockout Mutants Accumulate Increased Amounts of Circular RNAs.

Circular RNAs (circRNAs) are the products of the non-canonical splicing of pre-mRNAs. In contrast to humans and animals, our knowledge of the biogenesis and function of circRNAs in plants is very scarce. To identify proteins involved in plant circRNA generation, we characterized the transcriptomes of 18 Arabidopsis thaliana knockout mutants for genes related to splicing. The vast majority (>90%) of circRNAs were formed in more than one variant; only a small fraction of circRNAs was mutant-specific. Five times more circRNA types were identified in cbp80 and three times more in c2h2 mutants than in the wild-type. We also discovered that in cbp80, c2h2 and flk mutants, the accumulation of circRNAs was significantly increased. The increased accumulation of circular transcripts was not accompanied by corresponding changes in the accumulation of linear transcripts. Our results indicate that one of the roles of CBP80, C2H2 and FLK in splicing is to ensure the proper order of the exons. In the absence of one of the above-mentioned factors, the process might be altered, leading to the production of circular transcripts. This suggests that the transition toward circRNA production can be triggered by factors sequestering these proteins. Consequently, the expression of linear transcripts might be regulated through circRNA production.

Expression Landscape of circRNAs in Arabidopsis thaliana Seedlings and Adult Tissues.

RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Identification of Altered Developmental Pathways in Human Juvenile HD iPSC With 71Q and 109Q Using Transcriptome Profiling.

In Huntington disease (HD) subtle symptoms in patients may occur years or even decades prior to diagnosis. HD changes at a molecular level may begin as early as in cells that are non-lineage committed such as stem cells or HD patients induced pluripotent stem cells (iPSCs) offering opportunity to enhance the understanding of the HD pathogenesis. In addition, juvenile HD non-linage committed cells were previously not directly investigated in detail by RNA-seq. In the present manuscript, we define the early HD and juvenile HD transcriptional alterations using 6 human HD iPS cell lines from two patients, one with 71 CAGs and one with 109 CAG repeats. We identified 107 (6 HD lines), 198 (3 HD71Q lines) and 217 (3 HD109Q lines) significantly dysregulated mRNAs in each comparison group. The analyses showed that many of dysregulated transcripts in HD109Q iPSC lines are involved in DNA damage response and apoptosis, such as CCND1, CDKN1A, TP53, BAX, TNFRSF10B, TNFRSF10C, TNFRSF10D, DDB2, PLCB1, PRKCQ, HSH2D, ZMAT3, PLK2, and RPS27L. Most of them were identified as downregulated and their proteins are direct interactors with TP53. HTT probably alters the level of several TP53 interactors influencing apoptosis. This may lead to accumulation of an excessive number of progenitor cells and potential disruption of cell differentiation and production of mature neurons. In addition, HTT effects on cell polarization also demonstrated in the analysis may result in a generation of incorrect progenitors. Bioinformatics analysis of transcripts dysregulated in HD71Q iPSC lines showed that several of them act as transcription regulators during the early multicellular stages of development, such as ZFP57, PIWIL2, HIST1H3C, and HIST1H2BB. Significant upregulation of most of these transcripts may lead to a global increase in expression level of genes involved in pathways critical for embryogenesis and early neural development. In addition, MS analysis revealed altered levels of TP53 and ZFP30 proteins reflecting the functional significance of dysregulated mRNA levels of these proteins which were associated with apoptosis and DNA binding. Our finding very well corresponds to the fact that mutation in the HTT gene may cause precocious neurogenesis and identifies pathways likely disrupted during development.

Effects of G-quadruplex topology on translational inhibition by tRNA fragments in mammalian and plant systems in vitro.

The folding of tRNA fragments (tRFs) into G-quadruplex structures and the implications of G-quadruplexes in translational inhibition have been studied mainly in mammalian systems. To increase our knowledge of these phenomena, we determined the influence of human and plant tRFs and model G-quadruplexes on translation in rabbit reticulocyte lysate and wheat germ extract. The efficiency of translational inhibition in the mammalian system was strongly associated with the type of G-quadruplex topology. In the plant system, the ability of a small RNA to adopt the G-quadruplex conformation was not sufficient to repress translation, indicating the importance of other structural determinants.