-
Methods (San Diego, Calif.) Jan 2008microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a... (Review)
Review
microRNAs (miRNAs) are challenging molecules to amplify by PCR because the miRNA precursor consists of a stable hairpin and the mature miRNA is roughly the size of a standard PCR primer. Despite these difficulties, successful real-time RT-PCR technologies have been developed to amplify and quantify both the precursor and mature microRNA. An overview of real-time PCR technologies developed by us to detect precursor and mature microRNAs is presented here. Protocols describe presentation of the data using relative (comparative C(T)) and absolute (standard curve) quantification. Real-time PCR assays were used to measure the time course of precursor and mature miR-155 expression in monocytes stimulated by lipopolysaccharide. Protocols are provided to configure the assays as low density PCR arrays for high throughput gene expression profiling. By profiling over 200 precursor and mature miRNAs in HL60 cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate, it was possible to identify miRNAs who's processing is regulated during differentiation. Real-time PCR has become the gold standard of nucleic acid quantification due to the specificity and sensitivity of the PCR. Technological advancements have allowed for quantification of miRNA that is of comparable quality to more traditional RNAs.
Topics: Animals; Base Sequence; Humans; MicroRNAs; Molecular Sequence Data; RNA Precursors; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 18158130
DOI: 10.1016/j.ymeth.2007.09.006 -
Biological Chemistry Jul 2022Transfer RNAs (tRNAs) are transcribed as precursor molecules that undergo several maturation steps before becoming functional for protein synthesis. One such processing... (Review)
Review
Transfer RNAs (tRNAs) are transcribed as precursor molecules that undergo several maturation steps before becoming functional for protein synthesis. One such processing mechanism is the enzyme-catalysed splicing of intron-containing pre-tRNAs. Eukaryotic tRNA splicing is an essential process since intron-containing tRNAs cannot fulfil their canonical function at the ribosome. Splicing of pre-tRNAs occurs in two steps: The introns are first excised by a tRNA-splicing endonuclease and the exons are subsequently sealed by an RNA ligase. An intriguing complexity has emerged from newly identified tRNA splicing factors and their interplay with other RNA processing pathways during the past few years. This review summarises our current understanding of eukaryotic tRNA splicing and the underlying enzyme machinery. We highlight recent structural advances and how they have shaped our mechanistic understanding of tRNA splicing in eukaryotic cells. A special focus lies on biochemically distinct strategies for exon-exon ligation in fungi versus metazoans.
Topics: Eukaryotic Cells; Goals; Introns; RNA Precursors; RNA Splicing; RNA, Transfer; Saccharomyces cerevisiae
PubMed: 35621519
DOI: 10.1515/hsz-2021-0402 -
The Journal of Biological Chemistry May 1998
Review
Topics: Amino Acid Sequence; Archaea; Endonucleases; Molecular Sequence Data; RNA Precursors; RNA Splicing; RNA, Transfer; Saccharomyces cerevisiae; Sequence Homology, Amino Acid
PubMed: 9582290
DOI: 10.1074/jbc.273.21.12685 -
Seminars in Cell & Developmental Biology Dec 2015A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open... (Review)
Review
A majority of messenger RNA precursors (pre-mRNAs) in the higher eukaryotes undergo alternative splicing to generate more than one mature product. By targeting the open reading frame region this process increases diversity of protein isoforms beyond the nominal coding capacity of the genome. However, alternative splicing also frequently controls output levels and spatiotemporal features of cellular and organismal gene expression programs. Here we discuss how these non-coding functions of alternative splicing contribute to development through regulation of mRNA stability, translational efficiency and cellular localization.
Topics: Alternative Splicing; Animals; Evolution, Molecular; Gene Expression Regulation, Developmental; Humans; Models, Genetic; Morphogenesis; Protein Biosynthesis; RNA Precursors; RNA Stability
PubMed: 26493705
DOI: 10.1016/j.semcdb.2015.10.018 -
Molekuliarnaia Biologiia 2015RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing,... (Review)
Review
RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing, polyadenylation, mRNA stabilization, mRNA export from the nucleus to the cytoplasm, mRNA localization, and translation. RBPs regulate these processes primarily by binding to specific sequence elements in newly synthesized or mature transcripts. While many RPBs are known to recognize certain nucleotide sequences in RNA, information is insufficient for others. In particular, RBPs often compete for RNA binding or interact with RNA cooperatively. Hence, it is of importance to study the RNA-protein interactions in vivo. Numerous methods have been developed to identify the target nucleotide sequences of RBPs. The methods include the electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP) and its variants, and measurement of the level for newly synthesized transcripts. Each of the methods has its limitation, and several methods supplementing each other should be employed in order to detect the RNA sequence to which a protein binds.
Topics: Animals; Binding Sites; Electrophoretic Mobility Shift Assay; Gene Expression Regulation; Humans; Immunoprecipitation; Protein Binding; RNA Precursors; RNA, Messenger; RNA-Binding Proteins; SELEX Aptamer Technique; Sequence Analysis, RNA
PubMed: 26107901
DOI: 10.7868/S0026898415020111 -
Nucleic Acids Research Nov 2022Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity....
Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.
Topics: Humans; RNA Precursors; Histones; Polyadenylation; mRNA Cleavage and Polyadenylation Factors; RNA, Messenger
PubMed: 36447390
DOI: 10.1093/nar/gkac878 -
Biochimica Et Biophysica Acta. Gene... 2019RNA splicing, the process through which intervening segments of noncoding RNA (introns) are excised from pre-mRNAs to allow for the formation of a mature mRNA product,... (Review)
Review
RNA splicing, the process through which intervening segments of noncoding RNA (introns) are excised from pre-mRNAs to allow for the formation of a mature mRNA product, has long been appreciated for its capacity to add complexity to eukaryotic proteomes. However, evidence suggests that the utility of this process extends beyond protein output and provides cells with a dynamic tool for gene regulation. In this review, we aim to highlight the role that intronic RNA plays in mediating specific splicing outcomes in pre-mRNA processing, as well as explore an emerging class of stable intronic sequences that have been observed to act in gene expression control. Building from underlying flexibility in both sequence and structure, intronic RNA provides mechanisms for post-transcriptional gene regulation that are amenable to the tissue and condition specific needs of eukaryotic cells. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.
Topics: Alternative Splicing; Animals; Gene Expression Regulation; Humans; Introns; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 31682938
DOI: 10.1016/j.bbagrm.2019.194439 -
Nature Communications Aug 2023Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets...
Cleavage and polyadenylation (CPA) is responsible for 3' end processing of eukaryotic poly(A)+ RNAs and preludes transcriptional termination. JTE-607, which targets CPSF-73, is the first known CPA inhibitor (CPAi) in mammalian cells. Here we show that JTE-607 perturbs gene expression through both transcriptional readthrough and alternative polyadenylation (APA). Sensitive genes are associated with features similar to those previously identified for PCF11 knockdown, underscoring a unified transcriptomic signature of CPAi. The degree of inhibition of an APA site by JTE-607 correlates with its usage level and, consistently, cells with elevated CPA activities, such as those with induced overexpression of FIP1, display greater transcriptomic disturbances when treated with JTE-607. Moreover, JTE-607 causes S phase crisis and is hence synergistic with inhibitors of DNA damage repair pathways. Together, our data reveal CPA activity and proliferation rate as determinants of CPAi-mediated cell death, raising the possibility of using CPAi as an adjunct therapy to suppress certain cancers.
Topics: Animals; Polyadenylation; RNA Precursors; mRNA Cleavage and Polyadenylation Factors; RNA, Messenger; Mammals; Neoplasms
PubMed: 37528120
DOI: 10.1038/s41467-023-39793-8 -
Cellular and Molecular Life Sciences :... Dec 2015The regulation of splice site (SS) usage is important for alternative pre-mRNA splicing and thus proper expression of protein isoforms in cells; its disruption causes... (Review)
Review
The regulation of splice site (SS) usage is important for alternative pre-mRNA splicing and thus proper expression of protein isoforms in cells; its disruption causes diseases. In recent years, an increasing number of novel regulatory elements have been found within or nearby the 3'SS in mammalian genes. The diverse elements recruit a repertoire of trans-acting factors or form secondary structures to regulate 3'SS usage, mostly at the early steps of spliceosome assembly. Their mechanisms of action mainly include: (1) competition between the factors for RNA elements, (2) steric hindrance between the factors, (3) direct interaction between the factors, (4) competition between two splice sites, or (5) local RNA secondary structures or longer range loops, according to the mode of protein/RNA interactions. Beyond the 3'SS, chromatin remodeling/transcription, posttranslational modifications of trans-acting factors and upstream signaling provide further layers of regulation. Evolutionarily, some of the 3'SS elements seem to have emerged in mammalian ancestors. Moreover, other possibilities of regulation such as that by non-coding RNA remain to be explored. It is thus likely that there are more diverse elements/factors and mechanisms that influence the choice of an intron end. The diverse regulation likely contributes to a more complex but refined transcriptome and proteome in mammals.
Topics: Alternative Splicing; Animals; Mammals; Models, Genetic; Nucleotide Motifs; Protein Processing, Post-Translational; RNA Precursors; RNA Splice Sites; RNA, Messenger
PubMed: 26370726
DOI: 10.1007/s00018-015-2037-5 -
International Journal of Molecular... Oct 2021Recent comprehensive genomic studies including single-cell RNA sequencing and characterization have revealed multiple processes by which protein-coding and noncoding RNA... (Review)
Review
Recent comprehensive genomic studies including single-cell RNA sequencing and characterization have revealed multiple processes by which protein-coding and noncoding RNA processing are dysregulated in many cancers. More specifically, the abnormal regulation of mRNA and precursor mRNA (pre-mRNA) processing, which includes the removal of introns by splicing, is frequently altered in tumors, producing multiple different isoforms and diversifying protein expression. These alterations in RNA processing result in numerous cancer-specific mRNAs and pathogenically spliced events that generate altered levels of normal proteins or proteins with new functions, leading to the activation of oncogenes or the inactivation of tumor suppressor genes. Abnormally spliced pre-mRNAs are also associated with resistance to cancer treatment, and certain cancers are highly sensitive to the pharmacological inhibition of splicing. The discovery of these alterations in RNA processing has not only provided new insights into cancer pathogenesis but identified novel therapeutic vulnerabilities and therapeutic opportunities in targeting these aberrations in various ways (e.g., small molecules, splice-switching oligonucleotides (SSOs), and protein therapies) to modulate alternative RNA splicing or other RNA processing and modification mechanisms. Some of these strategies are currently progressing toward clinical development or are already in clinical trials. Additionally, tumor-specific neoantigens produced from these pathogenically spliced events and other abnormal RNA processes provide a potentially extensive source of tumor-specific therapeutic antigens (TAs) for targeted cancer immunotherapy. Moreover, a better understanding of the molecular mechanisms associated with aberrant RNA processes and the biological impact they play might provide insights into cancer initiation, progression, and metastasis. Our goal is to highlight key alternative RNA splicing and processing mechanisms and their roles in cancer pathophysiology as well as emerging therapeutic alternative splicing targets in cancer, particularly in gastrointestinal (GI) malignancies.
Topics: Alternative Splicing; Antineoplastic Agents; Gastrointestinal Neoplasms; Humans; RNA Precursors; RNA, Neoplasm
PubMed: 34769221
DOI: 10.3390/ijms222111790