RNA Tagging Research Articles

Levels of 5′ RNA tags in plasma and buffy coat from EDTA blood increase with time

For biological sample banking it is important to precisely document sample treatment prior to extraction and storage. A major variable is the interval between blood sampling and subsequent processing and storage. We have determined the relationship between this time interval and frequency of 5' transcript tags. This study was designed to establish guidelines for collecting RNA from blood in prospective studies and ensure maximum availability of RNA analytes. Venous blood was collected from 40 healthy volunteers. Samples were processed immediately, 12, 24 and 36 h post collection and buffy coat and/or plasma removed. Total RNA was extracted and reverse transcribed, assays were optimized and levels of 5' RNA tags quantified by qPCR. Stably expressed reference genes were selected to examine 5' tags in plasma and buffy coat blood fractions. Whole blood was processed at various time points post collection to determine the affect on the presence and stability of 5' RNA tags. A significant increase (P < 0.05 to P < 0.001) in 5' RNA tags was observed at 12 h and up to 36 h in plasma and buffy coat samples isolated from EDTA blood which was maintained at 4 degrees C prior to processing when compared with plasma and buffy coat isolated from EDTA blood processed immediately. Over time 5' RNA tags increase in both plasma and buffy coat samples. It has been previously shown that removing cells from their normal environment produces cellular activation and up-regulation of pathways resulting in increased transcript expression. Positive correlation was observed between the time interval from sample collection to storage and amount of 5' transcript tags present. This increase could be due to white blood cells undergoing necrosis and lysis, or from RNA protected within apoptotic bodies. As 5' RNA tags were targeted using random primers for reverse transcription, even RNA partly degraded by RNases would have been detected.

λN-GFP: an RNA reporter system for live-cell imaging

We describe a GFP-based RNA reporter system (lambdaN-GFP) to visualize RNA molecules in live mammalian cells. It consists of GFP fused to an arginine-rich peptide derived from the phage lambda N protein, lambdaN22, which binds a unique minimal RNA motif and can be used to tag any RNA molecule. LambdaN-GFP uses a small and easy to engineer RNA tag, reducing the likelihood of perturbing the function of the tagged RNA molecule.

The colorectal microRNAome

MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.

Polarity withinpMandpEpromoted phage lambdacI-rexA-rexBtranscription and its suppression

The cI-rexA-rexB operon of bacteriophage lambda confers 2 phenotypes, Imm and Rex, to lysogenic cells. Immunity to homoimmune infecting lambda phage depends upon the CI repressor. Rex exclusion of T4rII mutants requires RexA and RexB proteins. Both Imm and Rex share temperature-sensitive conditional phenotypes when expressed from cI[Ts]857 but not from cI+ lambda prophage. Plasmids were made in which cI-rexA-rexB was transcribed from a non-lambda promoter, pTet. The cI857-rexA-rexB plasmid exhibited Ts conditional Rex and CI phenotypes; the cI+-rexA-rexB plasmid did not. Polarity was observed within cI-rexA-rexB transcription at sites in cI and rexA when CI was nonfunctional. Renaturation of the Ts CI857 repressor, allowing it to regain functionality, suppressed the polar effect on downstream transcription from the site in cI. The second strong polar effect near the distal end of rexA was observed for transcription initiated from pE. The introduction of a rho Ts mutation into the host genome suppressed both polar effects, as measured by its suppression of the conditional Rex phenotype. Strong suppression of the conditional Rex[Ts] phenotype was imparted by ssrA and clpP (polar for clpX) null mutations, suggesting that RexA or RexB proteins made under conditions of polarity are subject to 10Sa RNA tagging and ClpXP degradation.

A general method for rapid and nondenaturing purification of RNAs.

A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.

Improved full-length cDNA production based on RNA tagging by T4 DNA ligase.

Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5'-RACE-PCR run on a documented experimental system.

Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components.

An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P.

A ribozyme that ligates RNA to protein.

We have used a combination of in vitro selection and rational design to generate ribozymes that form a stable phosphoamide bond between the 5' terminus of an RNA and a specific polypeptide. This reaction differs from that of previously identified ribozymes, although the product is analogous to the enzyme-nucleotidyl intermediates isolated during the reactions of certain proteinaceous enzymes, such as guanyltransferase, DNA ligase, and RNA ligase. Comparative sequence analysis of the isolated ribozymes revealed that they share a compact secondary structure containing six stems arranged in a four-helix junction and branched pseudoknot. An optimized version of the ribozyme reacts with substrate-fusion proteins, allowing it to be used to attach RNA tags to proteins both in vitro and within bacterial cells, suggesting a simple way to tag a specific protein with amplifiable information.

Use of epitope tags for routine analysis of transgene expression.

Peptide and RNA epitope tags as tools for routine analysis of transgene expression and protein accumulation in transformed plant cell cultures was evaluated using three genes that encode very structurally and functionally different proteins. A T7 peptide was introduced at the amino- and carboxyl-termini of phosphinothricin-N-acetyl transferase and avidin and at the carboxyl-terminus of galactose oxidase. An RNA sequence that forms a higher order structure that is recognized by antibodies raised against the FLAG peptide was separately introduced into the 3' nontranslated region of these genes. Constructs were introduced into maize cell cultures using particle bombardment and transgene expression, protein accumulation, protein function and presence of the tags in RNA and/or protein as appropriate were evaluated in up to approximately 25 culture lines per construct. Results indicate that, while there will likely always be a need for some empirical evaluation of any tag-protein combination, introduction of the peptide tag at the amino-terminus was generally more successful than was incorporation at the carboxyl-terminus. RNA tags show promise for this purpose, but routine application will require development of a very sensitive immunoassay.

The Mouse Brain Transcriptome by SAGE: Differences in Gene Expression between P30 Brains of the Partial Trisomy 16 Mouse Model of Down Syndrome (Ts65Dn) and Normals

Trisomy 21, or Down syndrome (DS), is the most common genetic cause of mental retardation. Changes in the neuropathology, neurochemistry, neurophysiology, and neuropharmacology of DS patients' brains indicate that there is probably abnormal development and maintenance of central nervous system structure and function. The segmental trisomy mouse (Ts65Dn) is a model of DS that shows analogous neurobehavioral defects. We have studied the global gene expression profiles of normal and Ts65Dn male and normal female mice brains (P30) using the serial analysis of gene expression (SAGE) technique. From the combined sample we collected a total of 152,791 RNA tags and observed 45,856 unique tags in the mouse brain transcriptome. There are 14 ribosomal protein genes (nine underexpressed) among the 330 statistically significant differences between normal male and Ts65Dn male brains, which possibly implies abnormal ribosomal biogenesis in the development and maintenance of DS phenotypes. This study contributes to the establishment of a mouse brain transcriptome and provides the first overall analysis of the differences in gene expression in aneuploid versus normal mammalian brain cells.

RNA Regulatory Elements in the Genomes of Simple Retroviruses

The RNA genomes of simple retroviruses encode three genes (gag, pol,andenv) which are required for replication. In addition, there are at least three well-definedcis-acting structures which regulate important aspects of the viral life cycle. The packaging signal at the 5′ end of the RNA tags the genomic RNA for specific encapsidation into assembling virus. Since viral Env proteins are translated from spliced mRNAs,cis-acting splicing signals ensure that the proper ratio of spliced and unspliced viral RNAs is present in the infected cell. Finally,cis-acting elements at the 3′ end of the genome promote the export of unspliced RNAs from the nucleus for translation and encapsidation.

Tagging ribozyme reaction sites to follow trans–splicing in mammalian cells

In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.

Nonradioactive Labeling of RNA

A modified ribodinucleotide (named pSEEp) has been synthesized using commercially available components on a DNA synthesizer. The presence of a 3′-terminal primary amino group permits its coupling to a range of nonradioactive labels, exemplified here by fluorescein. The product of chemical derivatization of the parent dinucleotide is a good substrate for T4 RNA ligase-mediated coupling to RNA under very mild conditions; the target RNA is not itself subjected to chemical treatment. The well-defined product of this terminal labeling may be analyzed by, for example, the use of a fluorescence-based DNA sequencer. The applications documented serve to demonstrate the power of this approach suggesting that any procedure depending on the detection or targeted tagging of RNA may be adapted to using pSEEp and its derivatives.