Transcriptome-wide RNA interaction profiling reveals physical and functional targets of hnRNP L in human T cells - PubMed (original) (raw)

Transcriptome-wide RNA interaction profiling reveals physical and functional targets of hnRNP L in human T cells

Ganesh Shankarling et al. Mol Cell Biol. 2014 Jan.

Abstract

The RNA processing factor hnRNP L is required for T cell development and function. However, the spectrum of direct targets of hnRNP L activity in T cells has yet to be defined. In this study, we used cross-linking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) to identify the RNA binding sites of hnRNP L within the transcriptomes of human CD4(+) and cultured Jurkat T cells. We find that hnRNP L binds preferentially to transcripts encoding proteins involved in RNA processing and in Wnt and T cell receptor (TCR) signaling. This binding is largely conserved across both quiescent and activated T cells, in agreement with the critical role of hnRNP L throughout T cell biology. Importantly, based on the binding profile of hnRNP L, we validate numerous instances of hnRNP L-dependent alternative splicing of genes critical to T cell function. We further show that alternative exons with weak 5' splice site sequences specifically show a strong correlation between hnRNP L binding and hnRNP L-dependent splicing regulation. Together, these data provide the first transcriptome-wide analysis of the RNA targets of hnRNP L in lymphoid cells and add to the functional understanding of hnRNP L in human biology.

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Figures

FIG 1

FIG 1

Transcriptome-wide hnRNP L RNA interactions in primary human CD4+ T cells revealed by CLIP-seq. (a) Western blot of hnRNP L expression in resting and anti-CD3- and anti-CD28-stimulated human CD4+ T cells. Shown are both total expression (Total) and the efficiency of immunoprecipitation (IP) versus the protein remaining uncollected (Sup). Note that “Total” and “Sup” levels are 5% of IP levels. (b) Flow chart of analysis of CLIP-seq reads obtained from CD4+ cells from three independent donors. Each sample was analyzed before and after stimulation by anti-CD3 and anti-CD28. Data from resting CD4+ cells are shown in blue, while data from stimulated CD4+ cells are shown in red. Numbers of reads passing key filters in the analysis are shown, including the final number of binding sites defined within RefSeq transcripts in resting and stimulated human CD4+ cells (see Materials and Methods and Table S1 in the supplemental material for details). (c) Distribution of hnRNP L binding sites that map to each indicated feature of RefSeq mRNAs compared to the distribution of each feature in the total RefSeq transcriptome. (d and e) Z-scores for the enrichment of hexamers within binding sites in resting (d) and stimulated (e) cells were calculated by comparing observed hexamer frequencies within CLIP-defined hnRNP L binding sites to randomized binding profiles within bound transcripts. (Insets) The top 20 hexamers were aligned to generate sequence logos.

FIG 2

FIG 2

Transcriptome-wide hnRNP L RNA interaction profiles obtained in JSL1 T cells. (a) Six biological replicates of JSL1 T cells, representing triplicate samples of resting and PMA-stimulated cells, were subjected to CLIP-seq analysis. Data were processed by a pipeline identical to that used to analyze hnRNP L binding sites in CD4+ cells. (b) Nucleotides of each type of transcript feature were enumerated within hnRNP L binding sites for both resting and stimulated conditions. (c and d) Z-scores for the enrichment of hexamers within binding sites in resting (c) and stimulated (d) cells were calculated by comparing observed hexamer frequencies within CLIP-defined hnRNP L binding sites to randomized binding profiles within bound transcripts. (Insets) The top 20 hexamers were aligned to generate sequence logos.

FIG 3

FIG 3

CLIP-seq identifies common hnRNP L RNA interactions among primary and cultured T cells. (a) The percentages of overlapping nucleotides for different binding profiles were computed transcriptome-wide. The P value was ∼0 for all pairwise overlaps of the data compared to the overlap of 100 permutations of resting and stimulated CD4+ binding profiles randomized within bound transcripts (control). (b and c) Total binding sites in resting and stimulated binding profiles for CD4+ (b) and JSL1 (c) cells were classified as shared, biased, or condition specific as described in Materials and Methods and in Results. (d and e) UCSC Genome Browser view of CD45 exon 4 (d) or intron 6 from HNRNPL (e), showing binding profiles from four experimental conditions. Bars above the gene schematics indicate previously identified binding sites for hnRNP L (ESS1 in CD45 and CA region in HNRNPL).

FIG 4

FIG 4

HnRNP L regulates exon inclusion of transcripts important to T cell development and signaling. (a) Lysates from wild-type cells and from cells stably transfected with a lentivirus carrying shRNA targeted to hnRNP L (L-KD) were immunoblotted using antibodies against hnRNP L or tubulin to assess loading. (b to h) Representative RT-PCR analyses of the indicated genes. Gray and black boxes represent the variable and constitutive exons, respectively, while the black line represents introns. Blue boxes represent the hnRNP L binding sites (see Fig. S3 in the supplemental material for an expanded browser view of CLIP data). The percentages of inclusion (% Inc) of the variable exons are averages for at least three independent experiments; standard deviations (SD) are shown. (e) a1 and a2 represent mutually exclusive exons. (h) The dashed box denotes the poison exon, while % alt represents the percentage of inclusion of the poison exon relative to the three isoforms.

FIG 5

FIG 5

Binding of hnRNP L within exons represses exon inclusion. (a to e) Representative RT-PCR analyses of the indicated genes, as described in the legend to Fig. 4. The percentages of inclusion of the variable exons are averages from at least three independent experiments; standard deviations (SD) are shown. The asterisk in panel d indicates a nonspecific PCR product. See Fig. S4 in the supplemental material for an expanded browser view of CLIP data.

FIG 6

FIG 6

Validation of hnRNP L targets based on 5′ splice site strength. (a to h) Representative RT-PCR analysis of the indicated genes, as described in the legend to Fig. 4. 5′ss scores, as calculated by MaxEntScan, are shown for the alternative exons. The percentages of inclusion of the variable exons are averages from at least three independent experiments, and standard deviations (SD) are shown. See Fig. S5 in the supplemental material for an expanded browser view of CLIP data.

FIG 7

FIG 7

Condition-specific binding sites in JSL1 cells are not due to changes in transcript expression. (a and b) The difference in the gene expression level (expressed as the number of RNA-Seq reads per kilobase of transcript per million reads [RPKM]) between resting and stimulated JSL1 cells was calculated as log2(RPKM for stimulated cells/RPKM for resting cells) from preexisting data (24) and was plotted for all transcripts bearing resting-state-specific (a) or stimulated-state-specific (b) binding sites in JSL1 cells. (c) Hexamer enrichment for all resting-state-specific sites that are not in genes with a ≤−0.5 change in gene expression (as indicated by the gray bar in panel a). (Inset) Sequence logo generated by multiple alignment of the top 20 hexamers. (d) Hexamer enrichment for all stimulated-state-specific sites that are not in genes with a ≥0.5 change in gene expression (as indicated in panel b). (Inset) Sequence logo generated by multiple alignment of the top 20 hexamers.

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