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16 recordsLinked to original sources
PubMed2025

Northern Blotting: Protocols for Radioactive and Nonradioactive Detection of RNA.

Northern blotting is a common technique in RNA biology, allowing to detect and quantify RNAs of interest following separation by gel electrophoresis, transfer to a membrane, and hybridization of specific anti-complementary labelled probes. In this chapter, we describe our protocol for efficient RNA extraction from yeast, separation on agarose gel, and capillary transfer to a membrane. We provide two different methods for strand-specific detection of several types of RNAs using oligonucleotide probes, the first using radioactive 32P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes.

Blotting, Northern↗
PubMed2025

Dynamic Evolution of Poly-A Tail Lengths Visualized by RNAse H Assay and Northern Blot Using Nonradioactive Probes in Yeast.

Poly-A tail length dynamics have been extensively studied from yeast to human, mostly using reporter transcripts. Recent studies have been carried out genome-wide to determine the status of poly-A tails at steady state. However, poly-A tail measurement at equilibrium gives an overall length that reflects a mixture of the different poly-A tail sizes for a single transcript. New genome-scale techniques are emerging to estimate dynamic of poly-A tails lengths, but they are not yet routine and individual validation experiments are useful. In this chapter we describe a protocol for visualizing poly-A tail lengths following transcription inhibition for a reporter mRNA using denaturing poly-acrylamide gel electrophoresis and northern blot assay. This protocol is quick to set up, requires the purchase of only a few specific reagents, does not rely on radioactivity for RNA monitoring, and can be easily implemented in any molecular biology laboratory.

Poly A↗
PubMed2025

A general framework to over-express tRNA-derived fragments from their parental tRNAs in mammalian cells.

tRNA-derived fragments (tRFs), generated from the cleavage of mature or precursor tRNAs are a category of regulatory noncoding RNAs with diverse functions in physiological or pathophysiological conditions. Here we describe a framework for the over-expression of tRFs from their parental tRNAs in mammalian cells. The process involves bioinformatics analysis to identify specific tRNAs that produce the tRF, PCR amplification of corresponding tRNA genes, and insertion into expression vectors. Transfection is carried out in HEK293T cells and detection of tRFs is achieved through northern blotting and dual luciferase reporter assays. In the latter, a complementary sequence to the tRF of interest is inserted into the luciferase reporter. By observing the reduction in luciferase activity, we can validate the expression of tRFs. This method enables precise study of tRF functions and their roles in cellular processes.

Humans↗
PubMed2026

RNAi in the Rhizarian Phytopathogen Plasmodiophora brassicae: The Causal Agent of Clubroot Disease in Cruciferous Crops.

Although RNA interference (RNAi) is widespread and functionally important across eukaryotes, RNAi pathways are diverse or even lost in some lineages. Rhizaria represents a major and distinct eukaryotic supergroup that includes Plasmodiophora brassicae (Pb), the causal agent of cruciferous clubroot disease, yet RNAi in this lineage remains poorly understood. Here, we characterized an unusual RNAi pathway in Pb. Small RNA sequencing across five representative Pb life stages revealed abundant siRNAs and miRNAs characterized by a predominant 21-nt length, phased genomic distribution, 2-nt 3' overhangs, and a strong 5'-cytidine bias. Three Pb miRNAs were further validated by northern blotting and stem-loop RT-qPCR. Genome analysis identified two canonical AGO homologs, PbAGO1 and PbAGO2, but no Dicer homologs, except for an RNase III-containing Drosha-like protein, PbDRL. Functional analyses showed that PbAGO1 and PbAGO2 mediate gene silencing, whereas PbDRL is required for sRNA biogenesis. Further, the cell wall component chitin was identified from Pb zoosporangia during the early infection and RNAi interfering with its biosynthesis in transgenic plants of Arabidopsis and Brassica napus blocked Pb early infection and conferred broad-spectrum resistance. Our study uncovers an unusual RNAi pathway in Rhizaria and provides a promising strategy to control cruciferous clubroot disease.

Plasmodiophora brassicae↗
PubMed2025

Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells.

Transcription and RNA decay determine steady-state RNA levels in cells available for translation and RNA-mediated regulatory functions. Both processes can be assessed by various techniques, for majority, based on RNA labelling or chromatin immunoprecipitation, but require a high level of expertise. Here, we describe a cost-effective, fast, and simple protocol that enables the profiling of nascent and mature RNA in the cytoplasm, nucleoplasm, and chromatin through subcellular fractionation. The workflow can include α-amanitin inhibition of RNA Polymerase II to assess nascent RNAs as a proxy of transcriptional activity, or it can be used without this treatment to investigate distribution of partially processed or mature transcripts across distinct subcellular compartments. It is applicable for studying any of RNA biotypes, including small and long noncoding RNAs, mRNAs, and their splice variants, on both transcript-specific and transcriptome-wide scales. Nascent or mature RNAs isolated from each fraction can be further analyzed by any technique of choice (northern blot, reverse transcription, RNA sequencing).

Humans↗
PubMed2025

Methods for purification and characterization of nicked tRNAs.

While tRNA-derived fragments (tDRs) play important roles in gene expression regulation, it is technically challenging to distinguish bona fide tDRs from nicked tRNAs. This is because analytical techniques used to study RNA, such as northern blot, RT-qPCR or sequencing involve the use of denaturing reagents (e.g., phenol, formamide, urea) or physical procedures (e.g., heat) that convert nicked tRNAs into tRNA halves or other tDRs. In this chapter, we describe a protocol that enables the purification of nicked tRNAs under non-denaturing conditions that preserve their 3D structure. Purified nicked tRNAs can then be either enzymatically repaired into almost full-length tRNAs, or chromatographically separated from single-stranded tDRs before detection. These protocols will allow researchers to distinguish between structurally distinct but sequence identical tDRs and nicked tRNAs, disentangling their biological functions.

RNA, Transfer↗
PubMedDate not supplied

E. coli ribosomal RNA contains sequences homologous to insertion sequences IS1 and IS2.

The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. These elements can modulate gene expression, but it is not known whether they normally function in genetic control. To determine whether IS elements could exert control through specific RNA transcripts, we hybridised lambda NNC1857 r14 (carrying IS1) and pBR322 (carrying a portion of IS2) to Northern blots of E. coli RNA. Regions of homology between the IS elements and ribosomal RNA were observed. Computer analysis of reported nucleotide sequences detected large segments of homology between the IS elements and both 23S and 16S rRNA. Additional homologous sequences in phi X174 and a leader region of a ribosomal protein gene cluster were also detected. The homologous sequence between IS2 and 16S rTNA is the same sequence in phi X174 DNA which codes for the ends of the E and D gene and the start of J. The partial IS sequences may represent silent evolutionary remnants or they could modulate the expression of genes carrying these sequences.

Base Sequence↗
PubMed2000

OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9.

We have recently reported that OTOF underlies an autosomal recessive form of prelingual sensorineural deafness, DFNB9. The isolated 5-kb cDNA predicted a 1,230 amino acid (aa) C-terminus membrane-anchored cytosolic protein with three C2 domains. This protein belongs to a family of mammalian proteins sharing homology with the Caenorhabditis elegans fer-1. The two other known members of this family, dysferlin and myoferlin, both have six predicted C2 domains. By northern blot analysis, a 7-kb otoferlin mRNA could be detected in the human brain. We isolated the corresponding cDNA, which is expected to encode a 1,977-aa-long form of otoferlin with six C2 domains. A 7-kb cDNA derived from the murine orthologous gene, Otof, was also identified in the inner ear and the brain. The determination of the exon-intron structure of the human and murine genes showed that they are composed of 48 coding exons and extend approximately 90 kb and approximately 80 kb, respectively. Alternatively spliced transcripts could be detected that predict several long isoforms (six C2 domains) in humans and mice and short isoforms (three C2 domains) only in humans. Primers were designed to explore the first 19 OTOF exons, henceforth permitting exploration of the complete coding sequence of the gene in DFNB9 patients. In a southwestern Indian family affected by DFNB9, a mutation in the acceptor splice site of intron 8 was detected, which demonstrates that the long otoferlin isoforms are required for inner ear function.

Alternative Splicing↗
PubMed2000

Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23.

Genes causing nonsyndromic autosomal recessive deafness (DFNB12) and deafness associated with retinitis pigmentosa and vestibular dysfunction (USH1D) were previously mapped to overlapping regions of chromosome 10q21-q22. Seven highly consanguineous families segregating nonsyndromic autosomal recessive deafness were analyzed to refine the DFNB12 locus. In a single family, a critical region was defined between D10S1694 and D10S1737, approximately 0.55 cM apart. Eighteen candidate genes in the region were sequenced. Mutations in a novel cadherin-like gene, CDH23, were found both in families with DFNB12 and in families with USH1D. Six missense mutations were found in five families with DFNB12, and two nonsense and two frameshift mutations were found in four families with USH1D. A northern blot analysis of CDH23 showed a 9.5-kb transcript expressed primarily in the retina. CDH23 is also expressed in the cochlea, as is demonstrated by polymerase chain reaction amplification from cochlear cDNA.

Alleles↗
PubMed2001

Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F.

Human chromosome 10q21-22 harbors USH1F in a region of conserved synteny to mouse chromosome 10. This region of mouse chromosome 10 contains Pcdh15, encoding a protocadherin gene that is mutated in ames waltzer and causes deafness and vestibular dysfunction. Here we report two mutations of protocadherin 15 (PCDH15) found in two families segregating Usher syndrome type 1F. A Northern blot probed with the PCDH15 cytoplasmic domain showed expression in the retina, consistent with its pathogenetic role in the retinitis pigmentosa associated with USH1F.

Aged↗
PubMed2026

A Novel Long Noncoding RNA-LNC000133 Associated With Steroid-Induced Osteonecrosis of the Femoral Head Promotes Osteoblast Differentiation Through Bone Marrow Mesenchymal Stem Cells-Derived Exosomes Pathway: A Bioinformatics Validation and Detailed Mechanistic Study.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating disease caused by glucocorticoid abuse, characterized by complex pathogenesis and unclear molecular mechanisms. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) and their exosome-mediated signalling is a key contributor to SONFH, although the precise mechanisms remain to be elucidated. In this study, the differential expression profiles of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in exosomes derived from human BMSCs (hBMSCs) obtained from patients with SONFH compared to controls with femoral neck fractures were identified. Through next-generation sequencing, a novel lncRNA, LNC000133, associated with SONFH was discovered. Using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and competing endogenous RNA (ceRNA) network construction, the LNC000133/miR-362-5p/TGF-β3/SMAD3/BMP2 signalling axis was established. The definitive expression, localization and full-length sequence of LNC000133 in BMSCs were subsequently validated by Northern blot, quantitative real-time polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH) and rapid amplification of cDNA ends (RACE). Most notably, mechanistic studies demonstrated that LNC000133-modified BMSCs-derived exosomes were efficiently taken up by osteoblasts, which promoted proliferation and osteogenic differentiation by targeting the miR-362-5p/TGF-β3/SMAD3/BMP2 signalling pathway.

Humans↗
PubMed2004

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Alleles↗
PubMed2003

Identification and characterization of Prp45p and Prp46p, essential pre-mRNA splicing factors.

Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p. We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro. We demonstrate that Prp45p and Prp46p are spliceosome associated throughout the splicing process and both are essential for pre-mRNA splicing. Under nonsplicing conditions they also associate in coprecipitation assays with low levels of the U2, U5, and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a postsplicing snRNP complex.

Base Sequence↗
PubMed1994

Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma.

The RET proto-oncogene encodes a transmembrane receptor of the tyrosine kinase family and has frequently been found activated in human thyroid carcinomas of the papillary subtype. In most cases the activation consisted of the fusion of its tyrosine-kinase domain with the 5'-terminal region of a gene designated H4 or D10S170. We have named the resulting H4/RET chimeric oncogene RET/PTC. Another activated form of the RET oncogene has subsequently been found in a thyroid carcinoma and is now referred to as RET/PTC2. Here we report the identification and cloning of a novel rearranged version of the RET oncogene in a human thyroid papillary carcinoma. In this case the tyrosine-kinase domain of RET was fused to a sequence 790 bp long belonging to a new gene that we have named RFG (RET Fused Gene). This novel chimeric oncogene has been designated RET/PTC3. In order to have more insights into the function of RFG we have completely cloned and sequenced its cDNA. RFG predicted amino-acid sequence does not have any significant homology to any already known genes and is ubiquitously expressed in human and mouse tissues. Finally we provide evidence indicating that the rearrangement leading to the generation of RET/PTC3 occurred in vivo in the original tumor DNA.

Amino Acid Sequence↗
PubMed2024

Detection of mitochondrial tDRs in killifish embryos and other non-model organisms.

In recent years a diversity of small noncoding RNAs have been identified that originate from the mitochondrial genome. These mitosRNAs are often dominated by tRNA-derived small RNAs (mito-tDRs). Differential expression of mito-tDRs is associated with responses to stress. They also appear to be expressed differentially during development and their expression may be enriched in stress-tolerant animals. Very little is currently known about roles or modes of action of these sequences, although they are implicated in a diversity of processes such as cell cycle regulation, mRNA stability, regulation of ROS production, and import of proteins into the mitochondrion. To better understand the various roles these sequences may play, it is critical that we understand their diversity, cellular location, and the context for their expression. This protocol outlines the methodologies used to detect mitosRNAs, including mito-tDRs, in embryos and cells of the annual killifish Austrofundulus limnaeus. We highlight critical steps in the isolation of RNA, creation of sequencing libraries, bioinformatics processing of sequence data, and methods for validation of expression that support a robust discovery pipeline for mitosRNAs even from species with incomplete reference genome sequences.

Animals↗
PubMed2002

A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system.

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.

Amino Acid Sequence↗