scholarly journals An improved method of constructing degradome library suitable for sequencing using Illumina platform

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Yong-Fang Li ◽  
Miao Zhao ◽  
Menglei Wang ◽  
Junqiang Guo ◽  
Li Wang ◽  
...  
Keyword(s):  
Gene Family ◽  
Small Rna ◽  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Mirna Family ◽  
Mirna Biogenesis ◽  
Improved Method ◽  
Illumina Platform ◽  
Transcriptional Gene Regulation ◽  
Small Rna Library

Abstract Background Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5′ RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal. Results In this protocol, 3′ end of the 5′RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library. Conclusions Degradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.

scholarly journals Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

2020 ◽  
Author(s):  
Patricia Baldrich ◽  
Saleh Tamim ◽  
Sandra Mathioni ◽  
Blake Meyers
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Library Construction ◽  
Construction Methods ◽  
Wide Range ◽  
Secondary Sirnas ◽  
Two Stages ◽  
Material Library ◽  
Small Rna Library

ABSTRACTPlant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. High-throughput sequencing is used for the discovery and quantification of small RNAs. However, several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two stages of maize anthers to evaluate the performance of seven commercially-available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phasiRNAs, or tRNA-derived fragment. We also observed that ligation bias occurs at both ends of miRNAs and phasiRNAs, suggesting that the biased compositions observed in small RNA populations, including nonstoichiometric levels of phasiRNAs within a locus, may reflect a combination of biological and technical influences.

scholarly journals Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tao Fan ◽  
Yu-Zhen Zhao ◽  
Jing-Fang Yang ◽  
Qin-Lai Liu ◽  
Yuan Tian ◽  
...  
Keyword(s):  
Gene Family ◽  
Splice Site ◽  
Messenger Rna ◽  
Expression Patterns ◽  
Protein Structures ◽  
Single Copy ◽  
Central Component ◽  
Animal Kingdom ◽  
Functional Studies ◽  
U1 Snrnp

AbstractEukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.

scholarly journals Small RNA sequencing reveals distinct nuclear microRNAs in pig granulosa cells during ovarian follicle growth

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Derek Toms ◽  
Bo Pan ◽  
Yinshan Bai ◽  
Julang Li
Keyword(s):  
Granulosa Cells ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Ovarian Function ◽  
Ovarian Follicle ◽  
Small Rna Sequencing ◽  
Cell Fractionation ◽  
Steroid Synthesis ◽  
Non Coding Rna ◽  
Dna Binding Sites

AbstractNuclear small RNAs have emerged as an important subset of non-coding RNA species that are capable of regulating gene expression. A type of small RNA, microRNA (miRNA) have been shown to regulate development of the ovarian follicle via canonical targeting and translational repression. Little has been done to study these molecules at a subcellular level. Using cell fractionation and high throughput sequencing, we surveyed cytoplasmic and nuclear small RNA found in the granulosa cells of the pig ovarian antral preovulatory follicle. Bioinformatics analysis revealed a diverse network of small RNA that differ in their subcellular distribution and implied function. We identified predicted genomic DNA binding sites for nucleus-enriched miRNAs that may potentially be involved in transcriptional regulation. The small nucleolar RNA (snoRNA) SNORA73, known to be involved in steroid synthesis, was also found to be highly enriched in the cytoplasm, suggesting a role for snoRNA species in ovarian function. Taken together, these data provide an important resource to study the small RNAome in ovarian follicles and how they may impact fertility.

scholarly journals Extensive Analysis of miRNA Trimming and Tailing Indicates that AGO1 Has a Complex Role in miRNA Turnover

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 267
Author(s):  
Axel J. Giudicatti ◽  
Ariel H. Tomassi ◽  
Pablo A. Manavella ◽  
Agustin L. Arce
Keyword(s):  
Small Rna ◽  
Stress Responses ◽  
Cellular Localization ◽  
Mirna Biogenesis ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Extensive Analysis ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Argonaute 1

MicroRNAs are small regulatory RNAs involved in several processes in plants ranging from development and stress responses to defense against pathogens. In order to accomplish their molecular functions, miRNAs are methylated and loaded into one ARGONAUTE (AGO) protein, commonly known as AGO1, to stabilize and protect the molecule and to assemble a functional RNA-induced silencing complex (RISC). A specific machinery controls miRNA turnover to ensure the silencing release of targeted-genes in given circumstances. The trimming and tailing of miRNAs are fundamental modifications related to their turnover and, hence, to their action. In order to gain a better understanding of these modifications, we analyzed Arabidopsis thaliana small RNA sequencing data from a diversity of mutants, related to miRNA biogenesis, action, and turnover, and from different cellular fractions and immunoprecipitations. Besides confirming the effects of known players in these pathways, we found increased trimming and tailing in miRNA biogenesis mutants. More importantly, our analysis allowed us to reveal the importance of ARGONAUTE 1 (AGO1) loading, slicing activity, and cellular localization in trimming and tailing of miRNAs.

Plant MicroRNAs Responsive to Fungal Infection

2014 ◽  
Vol 941-944 ◽  
pp. 1141-1145 ◽  
Author(s):  
Hui Li Zhang ◽  
Lin Chen ◽  
Wen Na Li ◽  
Li Li Wang ◽  
Hong Yu Xie
Keyword(s):  
Fungal Pathogens ◽  
High Throughput Sequencing ◽  
Adaptive Immune System ◽  
Messenger Rnas ◽  
Disease Mechanism ◽  
Adaptive Immune ◽  
Alternative Approach ◽  
Crop Disease ◽  
Transcriptional Gene Regulation ◽  
And Control

MicroRNAs (miRNAs) are endogenous small RNAs transcribed from non-coding DNA, which have the capacity to base pair with the target mRNAs (messenger RNAs) to repress their translation or resulted in cleavage. We have paid much attention on the DNA and its coded proteins, the discovery of miRNAs as gene negatively regulators has led to a fundamental change in understanding of post-transcriptional gene regulation in plants. Fungal pathogens infection is the main cause of most economic crops diseases. Unlike humans, plants don’t evolved to have a adaptive immune system, they protect themselves with a mechanism consists of activation and response. Recently, high throughput sequencing validated that miRNA play a crucial role in plant-fungus interaction. A better understanding of miRNA-mediated disease mechanism in fungi should clarify the strategy of crop disease control. MiRNA-based manipulations as gene suppressors, such as artificial miRNAs, may emerge as a new alternative approach for the improvement of crops and control of crop disease.

MicroRNA annotation in plants: current status and challenges

2021 ◽  
Author(s):  
Yongxin Zhao ◽  
Zheng Kuang ◽  
Ying Wang ◽  
Lei Li ◽  
Xiaozeng Yang
Keyword(s):  
Small Rna ◽  
Signal To Noise Ratio ◽  
Current Status ◽  
Mirna Biogenesis ◽  
Plant Mirnas ◽  
Signal To Noise ◽  
Advantages And Disadvantages ◽  
Plant Mirna ◽  
History Of ◽  
Mirna Biogenesis Pathway

Abstract Last two decades, the studies on microRNAs (miRNAs) and the numbers of annotated miRNAs in plants and animals have surged. Herein, we reviewed the current progress and challenges of miRNA annotation in plants. Via the comparison of plant and animal miRNAs, we pinpointed out the difficulties on plant miRNA annotation and proposed potential solutions. In terms of recalling the history of methods and criteria in plant miRNA annotation, we detailed how the major progresses made and evolved. By collecting and categorizing bioinformatics tools for plant miRNA annotation, we surveyed their advantages and disadvantages, especially for ones with the principle of mimicking the miRNA biogenesis pathway by parsing deeply sequenced small RNA (sRNA) libraries. In addition, we summarized all available databases hosting plant miRNAs, and posted the potential optimization solutions such as how to increase the signal-to-noise ratio (SNR) in these databases. Finally, we discussed the challenges and perspectives of plant miRNA annotations, and indicated the possibilities offered by an all-in-one tool and platform according to the integration of artificial intelligence.

scholarly journals Unravelling the developmental and functional significance of an ancient Argonaute duplication

2020 ◽  
Author(s):  
Arie Fridrich ◽  
Vengamanaidu Modepalli ◽  
Magda Lewandowska ◽  
Reuven Aharoni ◽  
Yehu Moran
Keyword(s):  
Messenger Rna ◽  
High Throughput Sequencing ◽  
Genetic Manipulation ◽  
Sea Anemones ◽  
Differential Distribution ◽  
Animal Physiology ◽  
Rna Targets ◽  
Target Regulation ◽  
Bilaterian Animal ◽  
Almost All

AbstractmicroRNAs (miRNAs), base-pair to messenger RNA targets and guide Argonaute proteins to mediate their silencing. This target regulation is considered crucial for animal physiology and development. However, this notion is based exclusively on studies in bilaterians, which comprise almost all lab model animals. To fill this glaring phylogenetic gap, we characterized the functions of two Argonaute paralogs in the sea anemone Nematostella vectensis of the phylum Cnidaria, which is separated from bilaterians by ∼600 million years. Using genetic manipulation, Argonaute-immunoprecipitations and high-throughput sequencing we provide experimental evidence for the developmental importance of miRNAs in a non-bilaterian animal. Additionally, we uncover unexpected differential distribution of distinct miRNAs between the two Argonautes and the ability of one of them to load additional types of small RNAs. This enables us to postulate a novel model for evolution of miRNA precursors in sea anemones and their relatives, revealing alternative trajectories for metazoan miRNA evolution.

scholarly journals PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

2016 ◽  
Author(s):  
Alex M. Tamburino ◽  
Ebru Kaymak ◽  
Shaleen Shrestha ◽  
Amy D. Holdorf ◽  
Sean P. Ryder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Small Rna ◽  
Rna Binding ◽  
Mrna Translation ◽  
Single Experiment ◽  
Novel Interactions ◽  
Transcriptional Gene Regulation ◽  
Rna Interaction ◽  
Eukaryotic Genomes

SUMMARYInteractions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest – in a ‘gene-centered’ manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

Small RNA and transcriptome sequencing of a symptomatic peony plant reveals mixed infections with novel viruses

Plant Disease ◽  
2021 ◽  
Author(s):  
Anning Jia ◽  
Chenge Yan ◽  
Hang Yin ◽  
Rui Sun ◽  
Fei Xia ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Triple Gene Block ◽  
Field Investigation ◽  
Mixed Infections ◽  
Species Classification ◽  
Gene Block ◽  
Sequence Identity

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus (CNSV), apple stem grooving virus (ASGV), lychnis mottle virus (LycMoV), grapevine line pattern virus (GLPV), and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleaotides (nt) in length, comprising three open reading frames (ORFs) and shared 63.8–75.9% nucleotide sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57% and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length with five ORFs encoding the REP (ORF1), triple gene block (TGB, ORF2–4) and coat protein (CP, ORF5) proteins. Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus (GYMaV) within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

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