Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, likeO. sativa,Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs. By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, we have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs fromO. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from theNicotiana benthamianawhose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, our method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.

Bhendi yellow vein mosaic virus (BYVMV) belongs to the monopartite begomovirus associated with the β satellite. As a single-stranded DNA (ssDNA) virus, it should be amenable to transcriptional and post-transcriptional gene silencing (TGS and PTGS). Previously, we had demonstrated C2, C4 and βC1 to be having different levels of influence on PTGS. Hence in the present study, a series of experiments such as agroinfiltration, chop-polymerase chain reaction (PCR), quantitative PCR (qPCR) and bisulfite next generation sequencing (NGS) were designed to analyse the involvement of BYVMV proteins on DNA methylation suppression. From the preliminary studies, we concluded that BYVMV genes were responsible for TGS suppression and C2, C4 genes from BYVMV were selected for further studies. Agroinfiltration experiments with mutant C2 and C4 partial tandem repeat (PTR) constructs of BYVMV have confirmed the role of C2 and C4 in DNA methylation impairment. The protoplast replication assay has shown that C4 was not an impediment for viral DNA replication and subsequent agroinfiltration studies with the C4 mutant BYVMV PTR construct have revealed the involvement of C4 in viral DNA movement.

MicroRNAs (miRNAs) are crucial regulatory RNAs, originated from hairpin precursors. For the past decade, researchers have been focusing extensively on miRNA profiles in various plants. However, there have been few studies on the global profiling of precursor miRNAs (pre-miRNAs), even in model plants. Here, for the first time in a non-model plant—Abelmoschus esculentus with negligible genome information—we are reporting the global profiling to characterize the miRNAs and their associated pre-miRNAs by applying a next generation sequencing approach. Preliminarily, we performed small RNA (sRNA) sequencing with five biological replicates of leaf samples to attain 207,285,863 reads; data analysis using miRPlant revealed 128 known and 845 novel miRNA candidates. With the objective of seizing their associated hairpin precursors, we accomplished pre-miRNA sequencing to attain 83,269,844 reads. The paired end reads are merged and adaptor trimmed, and the resulting 40–241 nt (nucleotide) sequences were picked out for analysis by using perl scripts from the miRGrep tool and an in-house built shell script for Minimum Fold Energy Index (MFEI) calculation. Applying the stringent criteria of the Dicer cleavage pattern and the perfect stem loop structure, precursors for 57 known miRNAs of 15 families and 18 novel miRNAs were revealed. Quantitative Real Time (qRT) PCR was performed to determine the expression of selected miRNAs.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum annelida, in which the genomes of the marine oligochaete Capitella telata , and the freshwater leech Helobdella robusta have been sequenced and studied. The terrestrial annelids, in spite of their ecological relevance and unique biochemical repertoire, are represented by a single rough genome draft of Eisenia fetida (North American isolate), which suggested that extensive duplications have led to a large number of HOX genes in this annelid. Herein, we report the draft genome sequence of Eisenia fetida (Indian isolate), a terrestrial redworm widely used for vermicomposting assembled using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm, showed that many miRNA gene families have also undergone extensive duplications. Genes for several important proteins such as sialidases and neurotrophins were identified by RNA sequencing of tissue samples. We also used de novo assembled RNA-Seq data to identify genes that are differentially expressed during regeneration, both in the newly regenerating cells and in the adjacent tissue. Sox4, a master regulator of TGF-beta induced epithelial-mesenchymal transition was induced in the newly regenerated tissue. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. The metagenome of the worm, characterized using 16S rRNA sequencing, revealed the identity of several bacterial species that reside in the nephridia of the worm. Comparison of the bodywall and cocoon metagenomes showed exclusion of hereditary symbionts in the regenerated tissue. In summary, we present extensive genome, transcriptome and metagenome data to establish the transcriptome and metagenome dynamics during regeneration.

MicroRNAs (miRNAs) are crucial regulatory RNAs, originated from hairpin precursors. For the past decade, researchers are focusing extensively on miRNA profiles in various plants. However studies on precursor miRNAs (pre-miRNAs) global profiling stay static even in model plants. Here, for the first time in a non-model plant, Abelmoschus esculentus with negligible genome information, we are reporting the global profiling to characterize the miRNAs and their associated pre-miRNAs by applying next generation sequencing approach. Preliminarily we performed sRNA (small RNA) sequencing with five biological replicates of leaf samples to attain 207,285,863 reads and data analysis using miRPlant keyed out 128 known and 845 novel miRNA candidates. With the objective to seize their associated hairpin precursors, we accomplished pre-miRNA sequencing to attain 83,269,844 reads. The paired end reads are merged, adaptor trimmed and the resulting 40-241 nt (nucleotide) sequences were picked out for analysis by using perl scripts from miRGrep tool and in-house built shell script for Minimum Fold Energy Index (MFEI) calculation. Applying stringent criteria of dicer cleavage pattern and perfect stem loop structure revealed precursors for 57 known miRNAs of 15 families and 18 novel miRNAs. Quantitative Real Time (qRT) PCR was performed to determine the expression of selected miRNAs.

Long considered to be active only in germline, PIWI/piRNA pathway is now known to play significant role in somatic cells, especially neurons. Nonetheless, so far there is no evidence for the presence of piRNAs in the neurosensory retina. In this study, we have uncovered 102 piRNAs in human retina and retinal pigment epithelium (RPE) by analysing RNA-seq data. The identified piRNAs were enriched with three motifs predicted to be involved in rRNA processing and sensory perception. Further, expression of piRNAs in donor eyes were assessed by qRT-PCR. Loss of piRNAs in HIWI2 knockdown ARPE19 cells downregulated targets implicated in intracellular transport (SNAREs and Rabs ), circadian clock ( TIMELESS ) and retinal degeneration ( LRPAP1 and RPGRIP1 ). Moreover, piRNAs were dysregulated under oxidative stress indicating their potential role in retinal pathology. Intriguingly, computational analysis revealed complete and partial seed sequence similarity between piR-62011 and sensory organ specific miR-183/96/182 cluster. Furthermore, the expression of retina enriched piR-62011 positively correlated with miR-182 in HIWI2 silenced Y79 cells. Thus, our data provides an evidence for the expression of piRNAs in human retina and RPE. Collectively, our work demonstrates that piRNAs dynamically regulate distinct molecular events in the maintenance of retinal homeostasis.