Reduction of Target Gene Expression by a Modified U1 snRNA

Beckley, S. A.; Liu, P.; Stover, Mary Louise; Gunderson, Samuel; Lichtler, Alex; Rowe, David W.

doi:10.1128/mcb.21.8.2815-2825.2001

Cited by 41 publications

(73 citation statements)

References 80 publications

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“…2 A as an example]. Because the intron had no effect when inserted upstream, we conclude that U1 snRNA inhibits poly(A) tail addition only when base paired in the 3Ј terminal exon of the pre-mRNA, an idea consistent with the exon definition model (23) and already supported by our earlier work (12,13) and by data in Fig. 4C.…”

Section: Effects Of Increased Distance Multiple Binding Sites and Isupporting

confidence: 75%

“…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).…”

mentioning

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.…”

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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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: Effects Of Increased Distance Multiple Binding Sites and Isupporting

confidence: 75%

“…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%

See 3 more Smart Citations

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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Expanding RNA Silencing Approaches by U1 Adaptors

Grünweller

Hartmann

2009

ChemBioChem

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Silent partner: In a recent publication, Gunderson and co-workers described an approach to recruit the abundant U1 snRNP, a splicing subparticle, to the last exon of a pre-mRNA, in a sequence-specific manner. This mediates interaction of the U1-70K protein subunit with poly(A) polymerase, thus blocking polyadenylation and inducing pre-mRNA degradation. This novel promising strategy expands the repertoire of gene-silencing concepts.

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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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Reduction of Target Gene Expression by a Modified U1 snRNA

Cited by 41 publications

References 80 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

Expanding RNA Silencing Approaches by U1 Adaptors

The reciprocal regulation between splicing and 3′‐end processing

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