Dynamics of small interfering RNA - FRET based integrity measurements of siRNA in the cuvette & inside cells

RNAi ist ein bedeutendes Werkzeug zur Funktionsanalyse von Genen und hat großes Potential für den Einsatz in der Therapie. Obwohl effiziente Knockdowns in der Zellkultur erzielt werden, erweist sich eine in vivo Anwendung als schwierig. Die großen Hürden sind dabei der Transport der siRNA ins Zielgewebe und deren voranschreitende Degradierung.rnMarkierte siRNA kann sowohl zur eigenen Integritätsmessung als auch zur Lokalisierung verwendet werden. Zwei Farbstoffe an den jeweiligen 3’- bzw. -5’-Enden des Sense- bzw. Antisense-Stranges erzeugen ein robustes FRET-System (Hirsch et al. 2012). Das Verhältnis von FRET- zu Donor-Signal, das R/G-Ratio, dient zur sensitiven Klassifizierung des Integritätslevels einer siRNA Probe (Järve et al. 2007; Hirsch et al. 2011; Kim et al. 2010). Mit diesem System kann eine Degradierung von weniger als 5 % in der Küvette und in Zellen nachgewiesen werden.rnDie vorliegende Arbeit beschäftigt sich mit der Evaluierung von potentiellen FRET Farbstoffpaaren hinsichtlich deren Eignung für in vitro und in vivo Anwendung. Verschiedenste FRET-Paare, die das gesamte sichtbare Spektrum abdecken, wurden evaluiert und ermöglichen nun die Auswahl eines geeigneten Paares für die jeweilige Anwendung oder Kombination mit anderen Farbstoffen.rnMit Hilfe von Alexa555/Atto647N siRNA wurde ein erfolgreicher Einschluss von siRNA in Liposomen beobachtet. Eine anschließende Evaluierung der RNase-Protektion ergab für Liposomen, Nanohydrogele und kationische Peptide hervorragende protektive Eigenschaften. Basierend auf den Ergebnisse können diese und andere Transportsysteme nun für eine zelluläre Aufnahme optimiert werden.rnAtto488/Atto590 zeigte die besten Eigenschaften für Echtzeit-Integritätsmessungen in der Lebendzellmikroskopie. Verringerte Bleicheigenschaften und minimaler spektraler “Cross-Talk” ermöglichten es, transfizierte Zellen über einen Zeitraum von bis zu 8 Stunden zu beobachten. Mittels Atto488/Atto590 siRNA wurde die Einschleusung und Freisetzung in Zellen in Echtzeit untersucht. Dabei konnten Freisetzung und Verteilung in einzelnen Zellen beobachtet und analysiert werden. rnAuf eine anfängliche Phase mit hoher Freisetzungsrate folgte eine Phase mit geringerer Rate für den restlichen Beobachtungszeitraum. Die durchschnittliche Verweildauer im Zytosol betrug 24 und 58 Minuten, wobei zwischen lang- und kurzanhaltenden Ereignissen unterschieden werden konnte. Obwohl ein Import von siRNA in den Zellkern beobachtet wurde, konnte kein Schema bzw. genauer Zeitpunkt, in Bezug auf den Transfektionszeitraum für diese Ereignisse bestimmt werden. Die beobachteten Freisetzungsprozesse fanden sporadisch statt und Änderungen in der zellulären Verteilung geschahen innerhalb von wenigen Minuten. Einmal freigesetzte siRNA verschwand mit der Zeit wieder aus dem Zytosol und es blieben nur kleine Aggregate von siRNA mit immer noch geringer Integrität zurück.rn

Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus laevis oocytes. Sequence variations of the conserved cis-acting 5' and 3' elements of U RNA genes may partly explain the low abundance of U7 RNA.

A synthetic histone pre-mRNA-U7 small nuclear RNA chimera undergoing cis cleavage in the cytoplasm of Xenopus oocytes.

The 3' processing of histone pre-mRNAs is a nuclear event in which the U7 small nuclear ribonucleoprotein (snRNP) participates as an essential trans-acting factor. We have constructed a chimeric histone-U7 RNA that when injected into the cytoplasm of Xenopus laevis oocytes assembles into a snRNP-like particle and becomes cleaved at the correct site(s). RNP assembly is a prerequisite for cleavage, but, since neither the RNA nor the RNP appreciably enter the nucleus, cleavage occurs mostly, if not exclusively, in the cytoplasm. Consistent with this, cleavage also occurs in enucleated oocytes or in oocytes which have been depleted of U7 snRNPs. Thus all necessary components for cleavage must be present in the oocyte cytoplasm. The novel cleavage occurs in cis, involving only a single molecule of chimeric RNA with its associated proteins. This reaction is equally dependent upon base pairing interactions between histone spacer sequences and the 5'-end of the U7 moiety as the natural in trans reaction. These results imply that U7 is the only snRNP required for histone RNA processing. Moreover, the chimeric RNA is expected to be useful for further studies of the cleavage and assembly mechanisms of U7 snRNP.

Structures of four human pseudogenes for U7 small nuclear RNA.

Four U7 RNA-related sequences were isolated from a human genomic DNA library. None of the sequences completely match the published human U7 RNA sequence and all of them contain features typical of reverse-transcribed pseudogenes.

Inhibition of gene expression in human cells through small molecule-RNA interactions

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from d-and l-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC50 of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

Association of Nonribosomal Nucleolar Proteins in Ribonucleoprotein Complexes during Interphase and Mitosis

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin—a major nucleolar RNA-binding protein—contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Efficient 3′-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10

The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In Saccharomyces cerevisiae, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1/A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1/A2 sites.

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle–regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for χ2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.