Pervasive transcription of eukaryotic genomes results in expression of long non-coding RNAs (lncRNAs) most of which are poorly conserved in evolution and appear to be non-functional. However, some lncRNAs have been shown to perform specific functions, in particular, transcription regulation. Thousands of small open reading frames (smORFs, <100 codons) located on lncRNAs potentially might be translated into peptides or microproteins. We report a comprehensive analysis of the conservation and evolutionary trajectories of lncRNAs-smORFs from the moss Physcomitrium patens across transcriptomes of 479 plant species. Although thousands of smORFs are subject to substantial purifying selection, the majority of the smORFs appear to be evolutionary young and could represent a major pool for functional innovation. Using nanopore RNA sequencing, we show that, on average, the transcriptional level of conserved smORFs is higher than that of non-conserved smORFs. Proteomic analysis confirmed translation of 82 novel species-specific smORFs. Numerous conserved smORFs containing low complexity regions (LCRs) or transmembrane domains were identified, the biological functions of a selected LCR-smORF were demonstrated experimentally. Thus, microproteins encoded by smORFs are a major, functionally diverse component of the plant proteome.

Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in controlling the temperature regulation of plant-virus interactions are poorly characterised. To elucidate these further, we analysed the responses of potato plants cv Chicago to infection by potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), the latter of which is known to significantly increase plant susceptibility to PVY. Using RNAseq analysis, we showed that single and combined PVY and heat-stress treatments caused dramatic changes in gene expression, affecting the transcription of both protein-coding and non-coding RNAs. Among the newly identified genes responsive to PVY infection, we found genes encoding enzymes involved in the catalysis of polyamine formation and poly ADP-ribosylation. We also identified a range of novel non-coding RNAs which were differentially produced in response to single or combined PVY and heat stress, that consisted of antisense RNAs and RNAs with miRNA binding sites. Finally, to gain more insights into the potential role of alternative splicing and epitranscriptomic RNA methylation during combined stress conditions, direct RNA nanopore sequencing was performed. Our findings offer insights for future studies of functional links between virus infections and transcriptome reprogramming, RNA methylation and alternative splicing.

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Small open reading frames (<100 codons) that are located on long noncoding RNAs (lncRNAs) can encode functional microproteins. These microproteins are shown to play important roles in different cellular processes, such as cell proliferation, development and disease response [1] , [2] , [3] , [4] , [5] , [6] . However, there are only a few known lncRNA-encoded functional microproteins in plants. One such microprotein that was named PSEP3, was identified in the moss Physcomitrium patens by mass-spectrometry analysis. 57-aa PSEP3 contains Low Complexity Region (LCR) enriched with proline. We have previously shown that PSEP3 is translated in protonemata and gametophores of P. patens , and its knockout (KO line) or overexpression (OE line) affects protonemata growth [7] . We performed a quantitative proteomic analysis of the mutant lines with PSEP3 knockout and overexpression. 7-days old protonemata of wild type (WT line) and both mutant lines (KO and OE) were collected and used for iTRAQ-based proteomic experiments. LC-MS/MS data were processed using PEAKS Studio v.8 software with protein identification based on a Phytozome protein database. More analysis of PSEP3 effects on plant growth can be obtained in the paper published in Nucleic Acid Research [8] .

Nonsense-mediated RNA decay (NMD) mechanism controls the quality of eukaryotic mRNAs by degradation of aberrant transcripts with a premature stop codon (PTC) in a pioneer round of translation. Besides aberrant transcripts, up to 10% of normal mRNA transcripts can be regulated by NMD. As NMD machinery is associated with translation, this system takes part in proteome formation in eukaryotic cells [1 , 2] . However, no proteomic datasets of plants with deficient NMD system are currently available. Here, we provide an isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic dataset of the moss Physcomitrium patens smg1 knockout line. The kinase SMG1 is one of the key components of the NMD system in many organisms, including plants. 8-day old protonema of wild type and mutant lines was used for the iTRAQ experiment in three biological replicates. LC-MS/MS data were processed using PEAKS Studio v.8 Software with protein identification based on a Phytozome protein database. Differentially expressed protein groups up- and down-regulated in the smg1 knockout line were found in the resulting dataset. Presented data can improve our understanding of NMD functions in plants.