Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch

Liu, P.

doi:10.1093/nar/30.11.2329

Cited by 12 publications

(24 citation statements)

References 44 publications

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“…HeLa cells (14) and Ros 17͞2.8 cells (13) were passaged and cultured under standard conditions. For details, see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org.…”

Section: Methodsmentioning

confidence: 99%

“…Stable cell lines were produced as described (12,13) with neomycin as the selection reagent. Luciferase assays were done according to the manufacturer's directions.…”

Section: Methodsmentioning

confidence: 99%

“…Antibody dilutions were 1:2,000 for the anti-aryl sulfatase A (ASA) antibody (kindly provided by P. Manowitz, University of Medicine and Dentistry, Piscataway, NJ), 1:10,000 for the anti-GAPDH antibody (Chemicon), and 1:5,000 for the anti-osteocalcin antibody (Diagnostic Systems Laboratories, Webster, TX). Northern blotting was done as described (12,13) with probes generated by the random priming labeling method. The 900-nt Collagen 1a1 probe was derived from the C-terminal and 3Ј noncoding region of the rat Collagen 1a1 gene, which is the least conserved part between collagen genes.…”

Section: Methodsmentioning

confidence: 99%

“…A second example of such an on-off regulation is where the U1 small nuclear ribonucleoprotein (U1 snRNP) particle, normally involved in recognition of the 5Ј splice site sequence (5Јss) during pre-mRNA splicing, binds upstream of a poly(A) site inhibiting its usage (11)(12)(13)(14). Certain papillomaviruses use this type of regulation during early stages of infection to repress late gene expression via inhibition of the activity of the late poly(A) site by a U1 snRNP:5Јss complex (11,14).…”

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confidence: 99%

“…There are a number of examples in which expression of 5Ј end-mutated U1 snRNA leads to assembly of 5Ј end-mutated U1 snRNP able to base pair to target sequences thereby changing the pattern of pre-mRNA processing of the targeted gene. Examples include suppressor U1 snRNAs (i.e., U1 snRNPs) (18,19) that bind to and activate splicing at nonphysiological 5Ј splice sites as well as 5Ј end-mutated U1 snRNAs͞U1 snRNPs that bind the 3Ј UTR inhibiting expression of exogenous and stably expressed reporter genes (11)(12)(13). Here, for the first time, we use 5Ј end-mutated U1 snRNAs͞U1 snRNPs to inhibit expression of endogenous genes in mammalian cells (rat and human) and demonstrate that two U1 snRNAs base paired to a single target gene's pre-mRNA act synergistically, resulting in up to 700-fold Abbreviations: sn, small nuclear; RNP, ribonucleoprotein; 5Јss, 5Ј splice sequence; MT, mutant; SV40, simian virus 40; pSV, pPRL-SV40; SPA, synthetic cleavage and polyadenylation signal; OC, osteocalcin; COL, collagen 1a1; ASA, aryl sulfatase A.…”

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confidence: 99%

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Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA

Fortes

Cuevas

Guan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

127

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Reducing or eliminating expression of a given gene is likely to require multiple methods to ensure coverage of all of the genes in a given mammalian cell. We and others [Furth, P. A., Choe, W. T., Rex, J. H., Byrne, J. C., and Baker, C. C. (1994) Mol. Cell. Biol. 14, 5278 -5289] have previously shown that U1 small nuclear (sn) RNA, both natural or with 5 end mutations, can specifically inhibit reporter gene expression in mammalian cells. This inhibition occurs when the U1 snRNA 5 end base pairs near the polyadenylation signal of the reporter gene's pre-mRNA. This base pairing inhibits poly(A) tail addition, a key, nearly universal step in mRNA biosynthesis, resulting in degradation of the mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently or stably expressed 5 end-mutated U1 snRNA. Also, we determine the inhibitory mechanism and establish a set of rules to use this technique and to improve the efficiency of inhibition. Two U1 snRNAs base paired to a single pre-mRNA act synergistically, resulting in up to 700-fold inhibition of the expression of specific reporter genes and 25-fold inhibition of endogenous genes. Surprisingly, distance from the U1 snRNA binding site to the poly(A) signal is not critical for inhibition, instead the U1 snRNA must be targeted to the terminal exon of the pre-mRNA. This could reflect a disruption by the 5 end-mutated U1 snRNA of the definition of the terminal exon as described by the exon definition model. mutant U1 snRNAs ͉ gene expression inhibition ͉ polyadenylation inhibition

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Section: Methodsmentioning

confidence: 99%

“…Stable cell lines were produced as described (12,13) with neomycin as the selection reagent. Luciferase assays were done according to the manufacturer's directions.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA

Fortes

Cuevas

Guan

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

127

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The reciprocal regulation between splicing and 3′‐end processing

Kaida

2016

WIREs RNA

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Most eukaryotic precursor mRNAs are subjected to RNA processing events, including 5′‐end capping, splicing and 3′‐end processing. These processing events were historically studied independently; however, since the early 1990s tremendous efforts by many research groups have revealed that these processing factors interact with each other to control each other's functions. U1 snRNP and its components negatively regulate polyadenylation of precursor mRNAs. Importantly, this function is necessary for protecting the integrity of the transcriptome and for regulating gene length and the direction of transcription. In addition, physical and functional interactions occur between splicing factors and 3′‐end processing factors across the last exon. These interactions activate or inhibit splicing and 3′‐end processing depending on the context. Therefore, splicing and 3′‐end processing are reciprocally regulated in many ways through the complex protein–protein interaction network. Although interesting questions remain, future studies will illuminate the molecular mechanisms underlying the reciprocal regulation. WIREs RNA 2016, 7:499–511. doi: 10.1002/wrna.1348For further resources related to this article, please visit the WIREs website.

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U1 interference (U1i) for Antiviral Approaches

Blázquez

Fortes

2015

Advances in Experimental Medicine and Biology

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U1 snRNP (U1 small nuclear ribonucleoprotein) is an essential component of the splicing machinery. U1 snRNP also plays an additional role in 3'-end mRNA processing when it binds close to polyadenylation sites (PAS). Cotranscriptionally, U1 snRNP binding close to putative PAS prevents premature cleavage and polyadenylation and consequently safeguards pre-mRNA transcripts and defines promoter directionality. At the 3'-end of mRNAs, U1 snRNP binding to putative PAS may regulate mRNA length or inhibit polyadenylation and, therefore, gene expression. U1 interference (U1i) is a technique to inhibit gene expression based on the property of U1 snRNP to inhibit polyadenylation. It requires the expression of a modified U1 snRNP, which interacts with a target gene upstream of its PAS and inhibits target gene expression. U1i has been used to inhibit the expression of reporter or endogenous genes both in tissue culture and in animal models. In addition, U1i combination with RNA interference (RNAi), another RNA-based gene silencing technology, results in a synergistic increased inhibition. This is of special interest for antiviral therapy, where strong inhibitions may be required to decrease the expression of replicative viral RNAs and impact the replication cycle. Furthermore, the combination of U1i and RNAi-based inhibitors should prevent the appearance of viral variants resistant to the treatment and allows the dose of inhibitors to be decreased and a functional inhibition to be obtained with fewer off target effects. In fact, U1i has been used to inhibit the expression of HIV-1 and HBV, whose viral genomes express mRNAs that must be polyadenylated by the nuclear polyadenylation machinery. In the case of HBV, antiviral U1i has been combined with RNAi to demonstrate a strong inhibition of expression from HBV sequences in vivo. This shows that, although several aspects of U1i technology remain to be addressed, U1i and U1i combined with RNAi have great potential as antivirals.

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Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch

Cited by 12 publications

References 44 publications

Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA

Inhibiting expression of specific genes in mammalian cells with 5′ end-mutated U1 small nuclear RNAs targeted to terminal exons of pre-mRNA

The reciprocal regulation between splicing and 3′‐end processing

U1 interference (U1i) for Antiviral Approaches

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