The inhibitory efficacy of RNA POL III-expressed long hairpin RNAs targeted to untranslated regions of the HIV-1 5' long terminal repeat

Human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes persistent infection resulting in the demise of immune regulatory cells, and ensuing diseases associated with acquired immune deficiency syndrome (AIDS). Although current therapeutic modalities have had a significant impact on mortality rates, novel therapies are constantly needed to prevent the emergence of resistant viral variants that escape the effects of antivirals. RNA Interference (RNAi) is a promising therapeutic modality for the inhibition of HIV-1 RNAs. Traditionally, RNAi effector sequences include expressed short hairpin RNAs (shRNAs) or short interfering RNAs (siRNAs). Recently, expressed long hairpin RNAs (lhRNAs) have been used with the aim of generating multiple independent siRNAs, which simultaneously target different susceptible sites on HIV-1. Here, modified lhRNAs expressed from U6 RNA Pol III promoters were targeted to sites within the first transcribed sequences of the HIV-1 5' long terminal repeat (LTR) region. Both Tat-dependent and independent suppressive efficacy was demonstrated against subtype B and C reporter sequences; however, lhRNAs complementary to the TAR stem-loop were refractory to silencing. None of the lhRNAs induced an unwanted interferon response as measured by interferon beta levels. Silencing by the lhRNAs was not equal across the extent of its cognate sequence, with the greatest efficacy observed for sequences located at the base of the stem. Nevertheless, direct antireplicative activity was seen when targeting lhRNAs to a subtype B HIV clone pNL4-3 Luc and a subtype C wild-type HIV-1 strain, FV5. These data highlight distinct target loci within the 5' LTR of HIV-1 that are susceptible to lhRNA targeting, and may prove to have an important advantage over other RNAi target sites within HIV-1. Although lhRNAs themselves require further manipulation to improve their overall efficacy in generating multiple functioning siRNAs, they may prove useful in any combinatorial-based approach to treating HIV-1 infection.     

U1 snRNP increases RNA Pol II elongation rate to enable synthesis of long genes

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The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.     

An enhanced U6 promoter for synthesis of short hairpin RNA

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1^G93A) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.     

Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain.

The N-terminal RNA binding domain (RBD) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-nucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in turn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking results described here, and in more detail by a recently described co-crystal of this RBD with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking experiments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contrast to the wild-type sequence, which does not. This single uridine substitution in the 10-nucleotide loop is the site of cross-linking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RBD. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.     

Structure of a U.U pair within a conserved ribosomal RNA hairpin.

A conserved hairpin corresponding to nt 1057-1081 of large subunit rRNA (Escherichia coli numbering) is part of a domain targeted by antibiotics and ribosomal protein L11. The stem of the hairpin contains a U.U juxtaposition, found as either U.U or U.C in virtually all rRNA sequences. This hairpin has been synthesized and most of the aromatic and sugar protons were assigned by two-dimensional proton NMR. Distances and sugar puckers deduced from the NMR data were combined with restrained molecular dynamics calculations to deduce structural features of the hairpin. The two U residues are stacked in the helix, form one NH3-O4 hydrogen bond and require an extended backbone conformation (trans alpha and gamma) at one of the U nucleotides. The hairpin loop, UAGAAGC closed by a U-A pair, is the same size as tRNA anticodon loops, but not as well ordered.     

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.     

Comparison of chicken 7SK and U6 RNA polymerase III promoters for short hairpin RNA expression

Background  

RNA polymerase III (pol III) type 3 promoters such as U6 or 7SK are commonly used to express short-hairpin RNA (shRNA) effectors for RNA interference (RNAi). To extend the use of RNAi for studies of development using the chicken as a model system, we have developed a system for expressing shRNAs using the chicken 7SK (ch7SK) promoter.     

Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.     

Identification and comparison of the porcine H1, U6, and 7SK RNA polymerase III promoters for short hairpin RNA expression

Mammalian Genome - RNA polymerase III is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs. Thus, RNA polymerase III promoters are often...     

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.     

Complex role of the beta 2-beta 3 loop in the interaction of U1A with U1 hairpin II RNA

RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex.     

Molecular dynamics simulations of the complex between human U1A protein and hairpin II of U1 small nuclear RNA and of free RNA in solution.

RNA-protein interactions are essential to a wide range of biological processes. In this paper, a 0.6-ns molecular dynamics simulation of the sequence-specific interaction of human U1A protein with hairpin II of U1 snRNA in solution, together with a 1.2-ns simulation of the free RNA hairpin, is reported. Compared to the findings in the x-ray structure of the complex, most of the interactions remained stable. The nucleotide U8, one of the seven conserved nucleotides AUUGCAC in the loop region, was unusually flexible during the simulation, leading to a loss of direct contacts with the protein, in contrast to the situation in the x-ray structure. Instead the sugar-phosphate backbone of nucleotide C15 was found to form several interactions with the protein. Compared to the NMR structure of U1A protein complexed with the 3'-untranslated region of its own pre-mRNA, the protein core kept the same conformation, and in the two RNA molecules the conserved AUUGCAC of the loop and the closest CG base pair were located in very similar positions and orientations, and underwent very similar interactions with the protein. Therefore, a common sequence-specific interaction mechanism was suggested for the two RNA substrates to bind to the U1A protein. Conformational analysis of the RNA hairpin showed that the conformational changes of the RNA primarily occurred in the loop region, which is just involved in the sites of binding to the protein and in agreement with experimental observation. Both the loop and stem of the RNA became more ordered upon binding to the protein. It was also demonstrated that the molecular dynamics method could be successfully used to simulate the dynamical behavior of a large RNA-protein complex in aqueous solution, thus opening a path for the exploration of the complex biological processes involving RNA at a molecular level.     

Electron microscopy of U4/U6 snRNP reveals a Y-shaped U4 and U6 RNA containing domain protruding from the U4 core RNP

We describe the electron microscopic investigation of purified U4/U6 snRNPs from human and murine cells. The U4/U6 snRNP exhibits two morphological features, a main body approximately 8 nm in diameter and a peripheral filamentous domain, 7-10 nm long. Two lines of evidence suggest that the peripheral domain may consist of RNA and to contain U6 RNA as well as the 5' portion of U4 RNA. (a) Separation of the U4/U6 snRNA interaction regions from the core domains by site-directed cleavage of the U4 snRNA with RNase H gave filament-free, globular core snRNP structures. (b) By immuno and DNA-hybridization EM, both the 5' end of U4 and the 3' end of U6 snRNA were located at the distal region of the filamentous domain, furthest from the core. These results, together with our observation that the filamentous U4/U6 domain is often Y shaped, correlate strikingly with the consensus secondary structure proposed by Brow and Guthrie (1988. Nature (Lond.), 334:213-218), where U4 and U6 snRNA are base paired in such a way that two U4/U6 helices together with a stem/loop of U4 snRNA make up a Y-shaped U4/U6 interaction domain.