Requirements for gene silencing mediated by U1 snRNA binding to a target sequence

Abstract

U1 interference (U1i) is a novel method to block gene expression. U1i requires expression of a 5′-end-mutated U1 snRNA designed to base pair to the 3′-terminal exon of the target gene's pre-mRNA that leads to inhibition of polyadenylation. Here, we show U1i is robust (≥95%) and a 10-nt target length is sufficient for good silencing. Surprisingly, longer U1 snRNAs, which could increase annealing to the target, fail to improve silencing. Extensive mutagenesis of the 10-bp U1 snRNA:target duplex shows that any single mismatch different from GU at positions 3–8, destroys silencing. However, mismatches within the other positions give partial silencing, suggesting that off-target inhibition could occur. The specificity of U1i may be enhanced, however, by the fact that silencing is impaired by RNA secondary structure or by splicing factors binding nearby, the latter mediated by Arginine-Serine (RS) domains. U1i inhibition can be reconstituted in vivo by tethering of RS domains of U1-70K and U2AF65. These results help to: (i) define good target sites for U1i; (ii) identify and understand natural cellular examples of U1i; (iii) clarify the contribution of hydrogen bonding to U1i and to U1 snRNP binding to 5′ splice sites and (iv) understand the mechanism of U1i.

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

“…To test the affinity of each RNA oligonucleotide for U1snRNP, we incubated the oligonucleotide pool with a purified fraction of human U1snRNP (Gunderson et al 1998;Abad et al 2008). After verifying that the U1snRNP prep was competent to bind the control BPV 59ss probes, we adjusted the ratio of probe to protein such that z5% of the oligonucleotide pool was shifted ( Fig.…”

“…Electrophoresis was for 3 h at 20 V/cm. The purification of U1snRNP (judged to be >98% pure) from HeLa cells is described in Abad et al (2008).…”

“…The binding of some factors such as the snRNPs is thought to be well understood. Through a mechanism of RNA:RNA base-pairing, U1snRNP predominantly recognizes the 59ss (Mount et al 1983), but there have also been reports of binding outside the 59ss (Puig et al 1999;McCullough and Berget 2000), and also reports of U1snRNP performing nonsplicing functions (Furth et al 1994;Abad et al 2008). In addition to cooperation between the factors that recognize these core elements, there are a variety of other factors that act as splicing activators and repressors by binding pre-mRNA to recruit or to modulate the recruitment of other components of the splicing machinery to the splice sites.…”

U1 interference (U1i) for Antiviral Approaches

“…Thus, single mutations abrogate U1in binding and therefore target inhibition [ 24 ]. In vivo, the exchange of the two central nucleotides of a functional U1in binding site inhibits gene silencing [ 30 ].…”

RNA-interference-based Gene Therapy Approaches to HIV Type-1 Treatment: Tackling the Hurdles from Bench to Bedside

“…A new addition to this arsenal is an antisense molecule that can elicit transcriptional gene silencing at the viral LTR promoter [115]. Another novel technique is RNAu, which is based on expression of a modified U1 small nuclear RNA (snRNA) [116]. The 5′ nucleotides 2-11 of U1 are replaced to allow base pairing with a 10-nucleotide target in the 3′-terminal exon of the gene of interest.…”