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U1 Adaptors: a new gene silencing technology

ABSTRACT: The goal of the microarray experiment was to do a head-to-head comparison of the U1 Adaptor technology with siRNA in terms of specificity at the genome-wide level. U1 Adaptors represent a novel gene silencing method that employs a mechanism of action distinct from antisense and RNA interference (RNAi). The U1 Adaptor is a bifunctional oligonucleotide having a “Target Domain” that is complementary to a site in the target gene's terminal exon and a “U1 Domain” that binds to the U1 small nuclear RNA (snRNA) component of the U1 small nuclear ribonucleoprotein (U1 snRNP) splicing factor. Tethering of U1 snRNP to the target pre-mRNA inhibits 3' end processing (i.e., polyA tail addition) leading to degradation of that RNA species within the nucleus thereby reducing mRNA levels. We demonstrate that U1 Adaptors can specifically inhibit both reporter and endogenous genes. Further, targeting the same gene either with multiple U1 Adaptors or with U1 Adaptors and small interfering RNAs (siRNAs), strongly enhances gene silencing, the latter as predicted from their distinct mechanisms of action. Such combinatorial targeting requires lower amounts of oligonucleotides to achieve potent silencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE14434 | GEO | 2009/02/08

SECONDARY ACCESSION(S): PRJNA111767

REPOSITORIES: GEO

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Platform comparison of U1 Adaptors - a new gene silencing technology vs. siRNA

Project description:The goal of the microarray experiment was to do a head-to-head comparison of the U1 Adaptor technology with siRNA in terms of specificity at the genome-wide level. U1 Adaptors represent a novel gene silencing method that employs a mechanism of action distinct from antisense and RNA interference (RNAi). The U1 Adaptor is a bifunctional oligonucleotide having a âTarget Domainâ that is complementary to a site in the target gene's terminal exon and a âU1 Domainâ that binds to the U1 small nuclear RNA (snRNA) component of the U1 small nuclear ribonucleoprotein (U1 snRNP) splicing factor. Tethering of U1 snRNP to the target pre-mRNA inhibits 3' end processing (i.e., polyA tail addition) leading to degradation of that RNA species within the nucleus thereby reducing mRNA levels. We demonstrate that U1 Adaptors can specifically inhibit both reporter and endogenous genes. Further, targeting the same gene either with multiple U1 Adaptors or with U1 Adaptors and small interfering RNAs (siRNAs), strongly enhances gene silencing, the latter as predicted from their distinct mechanisms of action. Such combinatorial targeting requires lower amounts of oligonucleotides to achieve potent silencing. Experiment Overall Design: For each sample total RNA was prepared from 3 independent transfections and then were pooled and analyzed by QPCR and also by microarray.

2009-02-21 | E-GEOD-14434 | biostudies-arrayexpress

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.

2013-06-28 | E-GEOD-46433 | biostudies-arrayexpress

Homo sapiens

Project description:U1 Adaptors: a new gene silencing technology

| PRJNA111767 | ENA

Transcriptome changes in splicing inhibited cells

Project description:In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA–pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

2010-09-29 | GSE24179 | GEO

Alzheimer’s disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment [N40K mice RNA-seq]

Project description:Recent proteome and transcriptome profiling of Alzheimer's disease (AD) brains reveals RNA splicing dysfunction and U1 small nuclear ribonucleoprotein (snRNP) pathology containing U1-70K and its N-terminal 40-KDa fragment (N40K). Here we present a causative role of U1 snRNP dysfunction to neurodegeneration in primary neurons and transgenic mice (N40K-Tg), in which N40K expression exerts a dominant-negative effect to downregulate full-length U1-70K. N40K-Tg recapitulates N40K insolubility, neuronal degeneration, cognitive impairment, and erroneous splicing events in synaptic pathways.

2022-08-11 | GSE196871 | GEO

Alzheimer’s disease-associated U1 snRNP splicing dysfunction causes neuronal hyperexcitability and cognitive impairment [N40K neuron RNA-seq]

2022-08-11 | GSE196872 | GEO

Transcriptome analysis of U1 snRNA overexpression in human neurons

Project description:U1 snRNA is the small nuclear RNA that services as the backbone of U1 snRNP. Removing intron from pre-mRNA to produce mature mRNA is the core function of the U1 snRNA. As one of the most abundant small noncoding RNA in human cells – estimated to be in the region of 1x10^6 copies per human cell (Hela) – U1 snRNA level is much more abundant than the other snRNAs. However, the potential role of the different amounts of the snRNAs is still unknown. To study the function of U1 snRNA in human neurons, we used lentivirus vector to overexpression U1 snRNA. We analyzed the gene expression change and identified target genes of U1 snRNA overexpression. We uncovered a novel role of U1 snRNA in regulating gene expression in human neurons.

2020-01-01 | GSE124951 | GEO

Sequence specific recognition of U1 snRNP stem-loop 4 by the ubiquitin-like domain of U2 snRNP protein SF3A1 and its RGG motif

Project description:Cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) was used to study the interaction between the ubiquitin-like domain (UBL) of the protein SF3A1, a component of the U2 snRNP, with stem-loop 4 RNA (SL4) from the U1 snRNP. The complex was cross-linked and analysed both in isolation, and in broader context with components of the U1 snRNP. Cross-linking was performed using irradiation under 254 nm light, relying on the inherent reactivity of ribonucleotides.

2022-02-22 | PXD027189 | Pride

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

2013-06-28 | GSE46433 | GEO

iCLIP and NGS to map protein-RNA interactions for U1-snRNP proteins in Trypanosoma brucei

Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking

2014-05-16 | E-GEOD-43848 | biostudies-arrayexpress

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