scholarly journals Rolling Circle cDNA Synthesis Uncovers Circular RNA Splice Variants

2019 ◽  
Vol 20 (16) ◽  
pp. 3988 ◽  
Author(s):  
Aniruddha Das ◽  
Pranita K. Rout ◽  
Myriam Gorospe ◽  
Amaresh C. Panda
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Rolling Circle Amplification ◽  
Rnase H ◽  
Full Length ◽  
Accurate Identification ◽  
Junction Sequence ◽  
Rolling Circle ◽  
Forward Primer

High-throughput RNA sequencing and novel bioinformatic pipelines have identified thousands of circular (circ)RNAs containing backsplice junction sequences. However, circRNAs generated from multiple exons may contain different combinations of exons and/or introns arising from alternative splicing, while the backsplice junction sequence is the same. To be able to identify circRNA splice variants, we developed a method termed circRNA-Rolling Circle Amplification (circRNA-RCA). This method detects full-length circRNA sequences by performing reverse transcription (RT) in the absence of RNase H activity, followed by polymerase chain reaction (PCR) amplification of full-length circRNAs using a forward primer spanning the backsplice junction sequence and a reverse primer exactly upstream of the forward primer. By sequencing the PCR products, circRNA splice variants bearing the same backsplice junctions, which were otherwise only predicted computationally, could be experimentally validated. The splice variants were further predicted to associate with different subsets of target RNA-binding proteins and microRNAs, supporting the notion that different circRNA splice variants can have different biological impacts. In sum, the circRNA-RCA method allows the accurate identification of full-length circRNA sequences, offering unique insight into their individual function.

scholarly journals Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

2003 ◽  
Vol 2 (6) ◽  
pp. 1274-1287 ◽  
Author(s):  
Kaori Tanabe ◽  
Noriko Ito ◽  
Tomomi Wakuri ◽  
Fumiyo Ozoe ◽  
Makoto Umeda ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Small Region ◽  
Full Length ◽  
Rna Recognition Motifs ◽  
La Protein ◽  
C Terminus ◽  
Localization Signal ◽  
Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

scholarly journals SAM68: Signal Transduction and RNA Metabolism in Human Cancer

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Paola Frisone ◽  
Davide Pradella ◽  
Anna Di Matteo ◽  
Elisa Belloni ◽  
Claudia Ghigna ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signal Transduction ◽  
Nuclear Export ◽  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Human Cancer ◽  
Rna Metabolism ◽  
Regulatory Sequences

Alterations in expression and/or activity of splicing factors as well as mutations incis-acting splicing regulatory sequences contribute to cancer phenotypes. Genome-wide studies have revealed more than 15,000 tumor-associated splice variants derived from genes involved in almost every aspect of cancer cell biology, including proliferation, differentiation, cell cycle control, metabolism, apoptosis, motility, invasion, and angiogenesis. In the past decades, several RNA binding proteins (RBPs) have been implicated in tumorigenesis. SAM68 (SRC associated in mitosis of 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family of RBPs. SAM68 is involved in several steps of mRNA metabolism, from transcription to alternative splicing and then to nuclear export. Moreover, SAM68 participates in signaling pathways associated with cell response to stimuli, cell cycle transitions, and viral infections. Recent evidence has linked this RBP to the onset and progression of different tumors, highlighting misregulation of SAM68-regulated splicing events as a key step in neoplastic transformation and tumor progression. Here we review recent studies on the role of SAM68 in splicing regulation and we discuss its contribution to aberrant pre-mRNA processing in cancer.

Rolling circle amplification and nanopore-based deep sequencing of full-length HBV genomes

2018 ◽  
Vol 68 ◽  
pp. S762 ◽  
Author(s):  
A. Mcnaughton ◽  
D. Bonsall ◽  
M.D. Cesare ◽  
A. Brown ◽  
D. Parkes ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Rolling Circle Amplification ◽  
Full Length ◽  
Rolling Circle

scholarly journals CELF2 regulates the species-specific alternative splicing of TREM2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Motoaki Yanaizu ◽  
Chika Washizu ◽  
Nobuyuki Nukina ◽  
Jun-ichi Satoh ◽  
Yoshihiro Kino
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Full Length ◽  
Splicing Regulation ◽  
Nonsense Mediated Mrna Decay ◽  
Specific Alternative ◽  
Species Specific ◽  
Human And Mouse ◽  
Paralogous Proteins

Abstract Genetic variations of TREM2 have been implicated as a risk factor of Alzheimer’s disease (AD). Recent studies suggest that the loss of TREM2 function compromises microglial responses to the accumulation of amyloid beta. Previously, we found that exon 3 of TREM2 is an alternative exon whose skipping leads to a reduction in full-length TREM2 protein by inducing nonsense-mediated mRNA decay. Here, we aimed to identify factors regulating TREM2 splicing. Using a panel of RNA-binding proteins, we found that exon 3 skipping of TREM2 was promoted by two paralogous proteins, CELF1 and CELF2, which were both linked previously with risk loci of AD. Although the overexpression of both CELF1 and CELF2 enhanced exon 3 skipping, only CELF2 reduced the expression of full-length TREM2 protein. Notably, the TREM2 ortholog in the green monkey, but not in the mouse, showed alternative splicing of exon 3 like human TREM2. Similarly, splicing regulation of exon 3 by CELF1/2 was found to be common to humans and monkeys. Using chimeric minigenes of human and mouse TREM2, we mapped a CELF-responsive sequence within intron 3 of human TREM2. Collectively, our results revealed a novel regulatory factor of TREM2 expression and highlighted a species-dependent difference of its regulation.

scholarly journals Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 4958-4967 ◽  
Author(s):  
Caroline Rivers ◽  
Andrea Flynn ◽  
Xiaoxiao Qian ◽  
Laura Matthews ◽  
Stafford Lightman ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Mammalian Species ◽  
Donor Site ◽  
Small Nuclear Rna ◽  
Endogenous Gene ◽  
The One

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression.

Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA–DNA chimaeras

2013 ◽  
Vol 449 (3) ◽  
pp. 719-728 ◽  
Author(s):  
Lydia Prongidi-Fix ◽  
Laure Schaeffer ◽  
Angelita Simonetti ◽  
Sharief Barends ◽  
Jean-François Ménétret ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Full Length ◽  
Molar Ratio ◽  
Detailed Knowledge ◽  
Histone H4 ◽  
Purification Method

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA–DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

scholarly journals Increased hnRNP K Expression Impacts Myelopoiesis By Altering RUNX1 Splicing

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3317-3317
Author(s):  
Sean M Post ◽  
Marisa J Aitken ◽  
Prerna Malaney ◽  
Xiaorui Zhang ◽  
Todd Link ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Fetal Liver ◽  
Full Length ◽  
Clinical Samples ◽  
Differentiation Potential ◽  
Hnrnp K ◽  
Mutational Status

Abstract Mutations in RNA binding proteins have been identified as pathogenic drivers in many hematological malignancies. However, in addition to mutational status, expression changes in RNA binding proteins likely impact disease processes. Through our studies, we identified that overexpression of hnRNP K (heterogeneous ribonucleoprotein K) -a poly(C)-RNA binding protein that governs the expression of numerous genes and transcripts- plays a pivotal role in myeloid malignancies. Using clinical samples, we determined that hnRNP K overexpression is a recurrent abnormality, occurring in nearly 30% of AML cases. Importantly, elevated hnRNP K levels associate with decreased overall survival (24.3 months versus 48.7 months; HR 1.9; 95% CI 1.3-2.7). However, the role of hnRNP K overexpression in AML remains unclear. To evaluate its putative oncogenic potential, we overexpressed hnRNP K in murine fetal liver cells (FLCs). Using colony formation assays (CFAs), we demonstrated that hnRNP K-overexpressing FLCs have an altered differentiation potential (increased number of immature (c-kit +Sca-1 +) and decreased number of mature myeloid (Gr1 +CD11b +) cells) and an increase in self-renewal capacity (increased number of colonies) (p=0.008). Mice transplanted with hnRNP K overexpressing FLCs had markedly shortened survival compared to empty vector controls, despite similar engraftment (median survival 8.1 weeks versus median not reached (HR 3.0, 95% CI 1.2 - 7.3, p=0.02). Significantly, extramedullary hematopoiesis was observed in the spleens and the hepatic parenchyma of mice transplanted with FLCs that overexpress hnRNP K. This resulted in disrupted splenic architecture and the presence of immature hematopoietic cells and cells of myeloid origin (CD117, CD14, and myeloperoxidase). Furthermore, analyses of the bone marrow revealed an increase in myeloid cells in hnRNP K transplanted mice. We next used unbiased and biochemical approaches to discover a direct interaction between hnRNP K and the RUNX1 transcript-a critical transcriptional factor often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1 (delExon6) , resulting in the generation of a functionally distinct isoform that is more stable than full-length RUNX1. RNA-Seq and reporter assays demonstrated that delExon6 has a unique transcriptional profile compared to full-length RUNX1, suggesting this spliced transcript may have a pathogenic role. To examine the functionality of delExon6, we performed CFAs. Here, we observed that delExon6 expression results in an increased proliferation potential that is mediated by hnRNP K's RNA binding activity. Together, these data establish hnRNP K as an oncogene in myeloid leukemia through its ability to directly bind the RUNX1 transcript, modify RUNX1 splicing, and subsequently alter its transcriptional activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals RNase H-dependent amplification improves the accuracy of rolling circle amplification combined with loop-mediated isothermal amplification (RCA-LAMP)

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11851
Author(s):  
Takema Hasegawa ◽  
Diana Hapsari ◽  
Hitoshi Iwahashi
Keyword(s):  
Detection Limit ◽  
Rolling Circle Amplification ◽  
Dna Amplification ◽  
Rnase H ◽  
Isothermal Amplification ◽  
Rolling Circle ◽  
Loop Mediated Isothermal Amplification ◽  
Specific Amplification ◽  
Negative Condition ◽  
Rnase H2

The hybrid method upon combining rolling circle amplification and loop-mediated isothermal amplification (RCA-LAMP) was developed to quantify very small amount of different type of RNAs, such as miRNAs. RCA-LAMP can help detect short sequences through padlock probe (PLP) circularization and exhibit powerful DNA amplification. However, one of the factors that determines the detection limit of RCA-LAMP is non-specific amplification. In this study, we improved the accuracy of RCA-LAMP through applying RNase H-dependent PCR (rhPCR) technology. In this method, the non-specific amplification was suppressed by using the rh primer, which is designed through blocking the modification at the 3′end to stop DNA polymerase reaction and replacing the 6th DNA molecule from the end with RNA using RNase H2 enzyme. Traditional RCA-LAMP amplified the non-specific amplicons from linear PLP without a targeting reaction, while RCA-LAMP with rh primer and RNase H2 suppressed the non-specific amplification. Conversely, we identified the risk posed upon conducting PLP cyclization reaction using Splint R ligase in the RNA-targeting step that occurred even in the RNA-negative condition, which is another factor determining the detection limit of RCA-LAMP. Therefore, this study contributes in improving the accuracy of RNA quantification using RCA-LAMP.

Sign in / Sign up

Export Citation Format

Share Document