piRNAs in the human retina and retinal pigment epithelium reveal a potential role in intracellular trafficking and oxidative stress.

Long considered active only in the germline, the PIWI/piRNA pathway is now known to play a significant role in somatic cells, especially neurons. In this study, piRNAs were profiled in the human retina and retinal pigment epithelium (RPE). Furthermore, RNA immunoprecipitation with HIWI2 (PIWIL4) in ARPE19 cells yielded 261 piRNAs, and the expression of selective piRNAs in donor eyes was assessed by qRT-PCR. Intriguingly, computational analysis revealed complete and partial seed sequence similarity between piR-hsa-26131 and the sensory organ specific miR-183/96/182 cluster. Furthermore, the expression of retina-enriched piR-hsa-26131 was positively correlated with miR-182 in HIWI2-silenced Y79 cells. In addition, the lnc-ZNF169 sequence matched with two miRNAs of the let-7 family, and piRNAs, piR-hsa-11361 and piR-hsa-11360, which could modulate the regulatory network of retinal differentiation. Interestingly, we annotated four enriched motifs among the piRNAs and found that the piRNAs containing CACAATG and CTCATCAKYG motifs were snoRNA-derived piRNAs, which are significantly associated with developmental functions. However, piRNAs consisting of ACCACTANACCAC and AKCACGYTCSC motifs were mainly tRNA-derived fragments linked to stress response and sensory perception. Additionally, co-expression network analysis revealed cell cycle control, intracellular transport and stress response as the important biological functions regulated by piRNAs in the retina. Moreover, loss of piRNAs in HIWI2 knockdown ARPE19 confirmed altered expression of targets implicated in intracellular transport, circadian clock, and retinal degeneration. Moreover, piRNAs were dysregulated under oxidative stress conditions, indicating their potential role in retinal pathology. Therefore, we postulate that piRNAs, miRNAs, and lncRNAs might have a functional interplay during retinal development and functions to regulate retinal homeostasis.

High-quality reference transcriptome construction improves RNA-seq quantification in Oryza sativa indica.

The Reference Transcriptomic Dataset (RTD) is an accurate and comprehensive collection of transcripts originating from a given organism. It holds the key to precise transcript quantification and downstream analysis of differential expressions and regulations. Currently, transcriptome annotations for most crop plants are far from complete. For example, Oryza sativa indica (O. sativa indica) is reported to have 40,759 transcripts in the Ensembl database without alternative transcript isoforms and alternative splicing (AS) events. To generate a high-quality RTD, we conducted RNA sequencing of rice leaf samples collected at various time points during Rhizoctonia solani infection. The obtained reads were analyzed by adopting the recently developed computational analysis pipeline to assemble the RTD with increased transcript and AS diversity for O. sativa indica (IndicaRTD). After stringent quality filtering, the newly constructed transcriptome annotation was comprised of 122,968 non-redundant transcripts from 53,695 genes. This study identified many novel transcripts compared to Ensembl deposited data that are important for regulating molecular and physiological processes in the plant system. Currently, the assembled IndicaRTD must allow fast quantification of transcript and gene expression with high precision.

Cost-Effective Transcriptome-Wide Profiling of Circular RNAs by the Improved-tdMDA-NGS Method.

Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5'-3' backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from Oryza sativa subsp. Indica. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.

An Insight Into Pentatricopeptide-Mediated Chloroplast Necrosis via microRNA395a During Rhizoctonia solani Infection.

Sheath blight (ShB) disease, caused by Rhizoctonia solani, is one of the major biotic stress-oriented diseases that adversely affect the rice productivity worldwide. However, the regulatory mechanisms are not understood yet comprehensively. In the current study, we had investigated the potential roles of miRNAs in economically important indica rice variety Pusa Basmati-1 upon R. solani infection by carrying out in-depth, high-throughput small RNA sequencing with a total data size of 435 million paired-end raw reads from rice leaf RNA samples collected at different time points. Detailed data analysis revealed a total of 468 known mature miRNAs and 747 putative novel miRNAs across all the libraries. Target prediction and Gene Ontology functional analysis of these miRNAs were found to be unraveling various cellular, molecular, and biological functions by targeting various plant defense-related genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate the miRNAs and their putative target genes. Out of the selected miRNA-specific putative target genes, miR395a binding and its cleavage site on pentatricopeptide were determined by 5' RACE-PCR. It might be possible that R. solani instigated chloroplast degradation by modulating the pentatricopeptide which led to increased susceptibility to fungal infection.

The first complete genomic sequence of cardamom mosaic virus, a member of the genus Macluravirus (family Potyviridae).

The complete genome sequence of the KS isolate of cardamom mosaic virus (CdMV) was determined using transcriptome sequencing data from CdMV-infected Elettaria cardamomum as well as from overlapping cDNA clones made from RNA extracted from viral particles. The viral genome consists of 8249 nucleotides (nt) and encodes a large polyprotein of 2636 amino acids (aa). The polyprotein of CdMV shared 48.9%-67.4% aa sequence identity with other reported macluraviruses. Similar to the other members of genus Macluravirus, the genome of CdMV lacks the P1 coding region and the N-terminus of the HC-Pro coding region. The putative small open reading frame, PIPO, embedded within the P3 cistron, is preceded by a C(A)6 motif instead of G(A)6. Phylogenetic analysis based on the complete genome sequence aided the grouping of CdMV along with all other macluraviruses and showed that it is closely related to alpinia oxyphylla mosaic virus (AloMV). Among CdMV isolates, the KS isolate is most similar to the Appangala isolate based on disease symptoms and phylogeny.

Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification.

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, like O. 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 from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose 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.

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

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. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

Parentally inherited long non-coding RNA Cyrano is involved in zebrafish neurodevelopment.

Transfer of genetic material from parents to progeny via fusion of gametes is a way to ensure flow of information from one generation to the next. Apart from the genetic material, gametes provide a rich source of other factors such as RNA and proteins which can control traits of the embryo. Non-coding RNAs are not only carriers of regulatory information but can also encode memory of events of parental life. Here, we explore the possibility of parental inheritance of non-coding RNAs, especially long non-coding RNAs. Meta-analysis of RNA-seq data revealed several non-coding RNAs present in zebrafish oocyte, sperm and 2cell-stage. The embryo is transcriptionally silent at this stage, we rationalize that all the RNAs detectable at 2cell-stage are deposited either by sperm or oocyte or both and thus inherited. In the inherited pool, we noticed a conserved lncRNA, Cyrano previously known for zebrafish brain development. Knockdown of inherited Cyrano by miR-7 without changing zygotic Cyrano altered brain morphology at 24 hpf and 48 hpf. This defect could be partially rescued by injecting full length Cyrano lncRNA or a mutant resilient to knock-down by miR-7. In future, there is ample scope to check the possibility of inherited lncRNAs as carriers of memory of parental life events and building blocks that set up an initial platform for development.

Deciphering microRNAs and Their Associated Hairpin Precursors in a Non-Model Plant, Abelmoschus esculentus.

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.