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Identification of U1 snRNA associated proteins

ABSTRACT: In this project, we isolate U1 snRNA associated proteins in Arabidopsis thaliana. We used an antisense oligonucleotide specific for the U1 snRNA and analyzed associated proteins by mass spectrometry. As a control, the same experiments were performed with U2 snRNA- and lacZ-specifc antisense oligonucleotides.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Seedling

SUBMITTER: Irina Droste-Borel

LAB HEAD: Sascha Laubinger

PROVIDER: PXD045484 | Pride | 2023-12-04

REPOSITORIES: Pride

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			Action	DRS
	20180307_CO_0781SaLa_R17_pur_dil.raw	Raw
	20180307_CO_0781SaLa_R18_pur_dil.raw	Raw
	20180307_CO_0781SaLa_R19_pur_dil.raw	Raw
	20180307_CO_0781SaLa_R20_pur_dil.raw	Raw
	20180307_CO_0781SaLa_R21_pur_dil.raw	Raw

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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.

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Exogenous expression analysis of U1 snRNA mutation

Project description:To reveal the effect of U1 snRNA mutation, we performed exogenous expression analysis of U1 snRNA mutation. The pLKO.1-puro U6 sgRNA BfuAI stuffer lentiviral vector was modified by removing the internal U6 promoter. It was replaced by the U1 locus, including 393 bases of internal native U1 promoter, the U1 sequence, and 39 bases of 3’-flanking region. The r.3A>G mutation was introduced by site-directed mutagenesis. HEK-293T cells were transfected using Lipofectamine Plus with either pLKO.1-U1wt or pLKO.1-U1r.3a>g in duplicate. Messenger RNA library construction was performed based on oligo dT-based mRNA isolation using NEBNext® Poly(A) mRNA Magnetic Isolation Module. RNA Sequence was performed on NextSeq 550 using 100-bp paired-end mode.

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Project description:U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1p36. Imperfect copies of these ‘true’ (t)U1 snRNA genes, located on chromosome 1q12-21, were thought to be pseudogenes. However, many of these ‘variant’ (v)U1 snRNA genes produce fully-processed transcripts that are packaged into potentially functional particles. Using antisense oligonucleotides, we have achieved functional knockdown of a specific vU1 snRNA in HeLa cells and identified over 400 transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Total RNA from 4 biological repeats of vU1.8 snRNA and control knock-down were analysed using Affymetrix Human Exon ST 1.0 Array.

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Project description:In this study, we built a model of cytoplasmic aggregation of U1 snRNA in a rat cell line. By comparing the gene expression profiling of U1 snRNA accumulated cells with their controls, 916 (about 4% of 20,000) genes were identified significantly differentially expressed. These 595 over-expressed genes and 321 repressed genes were further analyzed by GO and KEGG pathway enrichment. As a result, three of 12 enriched pathways are well-known cancer pathways, while nine of them were associated to cancers in previous studies. These findings suggest that cytoplasmic U1 snRNA accumulation may cause cancers.

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