Splicing repression allows the gradual emergence of new Alu-exons in primate evolution
- UCL Institute of Neurology, United Kingdom
- MRC-Laboratory of Molecular Biology, United Kingdom
- Institute de Biologie de l’ENS (IBENS), CNRS UMR 8197, France
- University College London Genetics Institute, United Kingdom
- Goethe University Frankfurt, Germany
- Institute of Molecular Biology (IMB), Germany
Figures
Figure 1 with 4 supplements
Alu-exons are associated with reduced gene expression.
(A) Expression levels of genes containing a cryptic, alternative or constitutive Alu-exon across a panel of 16 human tissues. For each gene, the median expression level was calculated from the …
Figure 1—figure supplement 1
Alu-exon gene expression in various human tissues.
Expression levels of genes containing a cryptic, alternative or constitutive Alu-exons across a panel of human tissues. For each tissue, only expressed genes were selected (RPKM ≥ 1). Tissues are …
Figure 1—figure supplement 2
Quality control of cytoplasmic and nuclear RNA fractions.
(A) The efficiency of hnRNPC depletion was tested by semi-quantitative Western blot. For comparison, a dilution series of lysate from control cells was included. (B) Cytoplasmic and nuclear protein …
Figure 1—figure supplement 3
Alu-exon transcripts are frequently depleted from the cytoplasmic RNA pool.
The relative abundance of the Alu-exon transcripts of AGL, NUP133, TIMM23 and HELLS was measured in cytoplasmic and nuclear RNA of cells depleted of hnRNPC (siC #1 and #2), or control cells (‘no …
Figure 1—figure supplement 4
Cytoplasmic depletion of Alu-exon genes is not caused by a defect in mRNA export upon hnRNPC depletion.
(A) Percentage of reads mapping to snRNAs, introns or exons are shown for each sample. Examined were RNAseq samples of cytoplasmic and nuclear RNA of cells transfected with siRNAs for hnRNPC (siC #1 …
Figure 2 with 2 supplements
Alu-exons are a novel class of NMD substrates.
(A) Depletion of hnRNPC in total RNA led to significant expression changes in 601 protein-coding genes (p-value < 0.01), of which 166 contain an Alu-exon. Boxplot showing the distribution of …
Figure 2—figure supplement 1
Validation of disenabling NMD by UPF1 depletion.
(A) Quantitative RT-PCR was used to measure the abundance of three genes known to be NMD substrates, GADD45B, SMG5 and ATF3 (Chan et al., 2007), as well as HNRNPC and UPF1 for control. Each of the …
Figure 2—figure supplement 2
Expression analysis of NMD-sensitive and NMD-refractory Alu-exons.
The relative abundance of the Alu-exon transcript of six genes was measured by semi-quantitative RT-PCR in cytoplasmic RNA from control, hnRNPC- and UPF1-depleted cells. Shown are gel visualisations …
Figure 3 with 3 supplements
NMD-refractory transcripts are cytoplasmic and polysome-associated.
(A) Boxplot presenting the fold changes (log2) of Alu-exons as well as their two flanking introns and downstream and upstream exons upon hnRNPC or UPF1 depletion over control, as analysed by DEXSeq. …
Figure 3—figure supplement 1
Alu-exons with weak splice sites cause formation of intron transcripts.
A modified DEXSeq approach was used to test for significant intron retention (for details, see Methods) in hnRNPC/UPF1 co-depleted cells, covering specifically introns flanking Alu-exons (A and B) …
Figure 3—figure supplement 2
Cytoplasmic NMD-refractory transcripts.
(A) The relative abundance of the intron-retaining Alu transcripts of MCM3 and NUP133 were measured in cytoplasmic and nuclear RNA from cells depleted of hnRNPC (siC #1 and #2), or control cells …
Figure 3—figure supplement 3
Examples of intron-retaining Alu transcripts.
RNAseq data were visualised with IGV. Data were generated from polyA-selected RNA from cells depleted of hnRNPC (siC #1 and #2), UPF1 (siUPF1), or UPF1 and hnRNPC, or control cells (unspecific …
Figure 4 with 2 supplements
Alu-exon age correlates with loss of U-tracts and repression by hnRNPC.
All 798 Alu-exons validated by junction-spanning reads at either splice site were stratified by the number of substitutions from the Alu consensus sequence provided by RepeatMasker (Xiao et al., 2009…
-
Figure 4—source data 1
Percent exon inclusion of Alu-exons in different human tissues.
We list percent exon inclusion for each Alu-exon with sufficient coverage across the GTEx data (889 exons with at least 4000 junction-spanning reads across 8555 individual samples). In addition, the data table includes chromosome position of the exon, mean skipping counts, the maximum and minimum inclusion across the tissues, the maximum difference in exon inclusion across the tissues, strength of the 3' splice site. In human, the length of the longest U-tract, substitutions in the Alu elements and substitution group. If no junction-reads were detected in a tissue extending from the upstream directly to the downstream exon ('skipping junctions'), we report 100% exon inclusion in this tissue.
- https://doi.org/10.7554/eLife.19545.015
Figure 4—figure supplement 1
Repression of Alu-exons in dependence of U-tract length.
All Alu-exons in protein-coding genes that are validated by junction-spanning reads at both the 5' and the 3' splice site (SS) were selected. Boxplots showing the changes in exon abundance upon …
Figure 4—figure supplement 2
Features of Alu-exons grouped by substitution rate.
All Alu-exons in protein-coding genes that were validated by junction-spanning reads at both the 5' and the 3' splice site (SS; 798 exons in total, were stratified by the number of substitutions …
Figure 5 with 1 supplement
U-tracts of emerging Alu-exons lengthen in parallel to an abrupt gain of 3' splice site sequences.
For all known Alu-exons found in this study or annotated in UCSC, orthologous regions were identified in four other primate genomes and scanned for the presence of an Alu element and a 3' splice …
-
Figure 5—source data 1
List of Alu-exons across our datasets and UCSC annotation, including cross-species annotation.
We merged all Alu-exons identified from RNAseq data and annotated in UCSC (6731 exons), and filtered for non-overlapping exons. If any two exons overlapped, we retained the larger exon. This created a list of 6309 Alu-exons for which we selected the 3' splice site and mapped orthologues positions in four primate genomes. For each 3'splice site (if present), we predicted the splice site strength using MaxEntScan (Yeo and Burge, (2004) and searched for the longest U-tract within the Alu element. The table lists in order: Alu-exon co-ordinates (in hg19), and the annotation by UCSC as alternative or constitutive exon, if not present the exon is annotated as cryptic exon. Next, the furthest species in which we could identify the orthologous position, the exonisation group of the 3' splice site as defined in Figure 5 (see Materials and methods for details), the coordinates, repeat family, substitution group of the Alu element the exon arises from (in hg19), as well as the position, predicted strength of the 3' splice site and longest U-tract of the Alu element in human (hg19), in chimpanzee (panTro4), in gibbon (nomLeu1), in rhesus macaque (rheMac3) and in marmoset (calJac3). All 3' splice site positions start with the −2 position of the canonical 3' splice site (the AG nucleotide) consensus.
- https://doi.org/10.7554/eLife.19545.019
Figure 5—figure supplement 1
U-tract length of Alu-exons traced throughout primate orthologues.
We searched for the orthologues of all Alu-exons with a strong 3' splice site in human, either annotated in UCSC or described in this study (2867 Alu-exons in total). We used the following genomes: h…
Figure 6
Repressive elements co-evolve with splice site sequences at cryptic exons.
Alignment within primate genomes of Alu elements that contain human Alu-exon reveals a tight coupling between repressive U-tracts and the variation of 3' splice site strength. (A) As the strength of …
Author response image 1
(A) We selected the ~15% most lowly expressed genes across six human tissues (RPKM of 0.5-2, n=2,199), and compared their expression levels (RPKM) in other species. Data from Brawand et al. (Nature …
Additional files
-
Supplementary file 1
List of RT-PCR and RT-qPCR primers used in this study.
- https://doi.org/10.7554/eLife.19545.022
