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. 2013 Feb;23(2):281-91.
doi: 10.1101/gr.142968.112. Epub 2012 Oct 15.

Differentially Expressed, Variant U1 snRNAs Regulate Gene Expression in Human Cells

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Free PMC article

Differentially Expressed, Variant U1 snRNAs Regulate Gene Expression in Human Cells

Dawn O'Reilly et al. Genome Res. .
Free PMC article

Abstract

Human U1 small nuclear (sn)RNA, required for splicing of pre-mRNA, is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes, also located on chromosome 1 (1q12-21), were thought to be pseudogenes. However, many of these "variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells, we have identified global transcriptome changes following interrogation of the Affymetrix Human Exon ST 1.0 array. Our results indicate that this vU1 snRNA regulates expression of a subset of target genes at the level of pre-mRNA processing. This is the first indication that variant U1 snRNAs have a biological function in vivo. Furthermore, some vU1 snRNAs are packaged into unique ribonucleoproteins (RNPs), and many vU1 snRNA genes are differentially expressed in human embryonic stem cells (hESCs) and HeLa cells, suggesting developmental control of RNA processing through expression of different sets of vU1 snRNPs.

Figures

Figure 1.
Figure 1.
Annotation and sequence of the U1 snRNA class I pseudogenes. (A) Schematic of chromosome 1 illustrating the location of U1 snRNA genes (1p36) and the U1 snRNA class I pseudogenes (1q12-21). The size of each locus is shown in kilobases (kb). Each gene is represented by a vertical line and a number. Light and dark boxes on chromosome 1 refer to regions of high and low gene activity, respectively. (B) Alignment of the snRNA sequence of the U1 snRNA genes (U1.1–4) with the 21 vU1 snRNA genes. Paired vU1 snRNAs have identical sequences. Nonconserved bases are denoted with the letter code representing one of the four nucleotide bases. A dash indicates the presence of a corresponding base, and a gap indicates the absence of a base at that position. The bases are numbered, beginning with 1 for the first base of the U1 snRNA sequence. Important features of the U1 snRNA are boxed and indicated underneath the alignment: 5′ splice site recognition motif (5′ss), U1-70K protein binding region (U1-70K); U1-A protein binding region (U1-A); Sm binding site (Sm).
Figure 2.
Figure 2.
Several vU1 snRNA are transcribed in HeLa cells. (A) ChIP analysis of U1 snRNA and vU1 snRNA genes in HeLa cells with antibodies indicated at right. Error bars in this and subsequent figures represent standard error of at least three repeats. Location of the primers used is indicated on the diagram. The proximal sequence element (PSE), coding and 3′ box are specified by gray and black boxes, respectively, and start of transcription by an arrow. (B) Quantification of vU1 snRNA steady-state levels in HeLa cells, normalized to U1 snRNA. Only reads with two or more nucleotide differences from U1 snRNA were counted toward the vU1 snRNA levels. Experimental data from Fejes-Toth et al. (2009).
Figure 3.
Figure 3.
vU1 snRNA genes are up-regulated in hESCs. (A) ChIP analysis of the U1 snRNA and some vU1 snRNA genes in hESCs and HeLa cells with antibodies indicated at the right. (B) qRT-PCR analysis of nascent transcripts from the corresponding U1 snRNA and vU1 snRNA genes in HeLa and hESCs. (Rel. levels) The levels of vU1 snRNA nascent transcripts are expressed relative to U1 snRNA, normalized to 1.0 in each cell type.
Figure 4.
Figure 4.
Some vU1 snRNA genes are differentially regulated. Pol II ChIP analysis of the U1, vU1.2a, vU1.8, and vU1.18 snRNA genes in HeLa and hESCs, before and after differentiation to embryoid bodies (EB). The position of the primers is indicated in the schematics.
Figure 5.
Figure 5.
vU1 snRNAs are packaged into RNP complexes. qRT-PCR analysis of U1 snRNA and vU1 snRNA levels in HeLa (A) and hESC (B) extracts, before and after immunoprecipitation with anti-Sm (Y-12) antibody. The non-Sm-containing 7SK RNA level was used as a negative control. Location of the primers is indicated in A. The level of the U1 snRNA and vU1 snRNAs, enriched in the Sm-immunoprecipitate, is expressed relative to input at 100%. RT (reverse transcription) reactions without “–” or with “+” the addition of Superscript III. Note: Although vU1.15/vU1.16 snRNA was enriched in RNP complexes, molecular cloning, followed by sequencing, indicated that this variant was not properly processed at the 3′ end. The levels of the vU1 snRNAs, enriched in the Sm immunoprecipitates, relative to the U1 snRNA were estimated using a genomic DNA standard to normalize primer efficiency.
Figure 6.
Figure 6.
Altering vU1.8 snRNA levels cause global transcriptome changes in HeLa cells. (A) qRT-PCR analysis of vU1.8 and U1 snRNA levels in HeLa cells transfected with control or vU1.8 snRNA antisense oligonucleotides. (B) Average profile of intronic probe signal difference between vU1.8 snRNA knockdown and control cells across expressed nonoverlapping genes relative to the annotated transcription start sites. Each point represents an average of more than 100 probes in the corresponding region. (C) Validation of array results by qRT PCR, confirming the reduction in VAPA and PRPF4B pre-mRNA levels toward the 3′ end. Gene structures are illustrated above the graphs; horizontal and vertical lines indicate introns and exons, respectively. Location of primers is denoted as horizontal lines below the schematics. (Arrow) Cryptic poly(A) sites within the first intron of VAPA and PRPF4B genes. Sizes of genes are indicated in kilobases. (D) 3′ RACE, using nested PCR, on total RNA from HeLa cells transfected with control, vU1.8, or U1 snRNA antisense oligonucleotides was performed on the endogenous PRPF4B, VAPA, and NR3C transcripts to detect polyadenylated mRNAs. PCPA denotes a premature cleaved and polyadenylated mRNA product. (*) Mispriming of the oligo dT primer at an A-rich region. Primers targeting the endogenous beta-actin mRNA was used as a normalization control. Sequencing results of the PCPA products for the PRPF4B and VAPA gene are shown on the right. The putative poly(A) sites are indicated with black bordered boxes.

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