Global profiling of hnRNP A2/B1-RNA binding on chromatin highlights LncRNA interactions

Abstract

Long noncoding RNAs (lncRNAs) often carry out their functions through associations with adaptor proteins. We recently identified heterogeneous ribonucleoprotein (hnRNP) A2/B1 as an adaptor of the human HOTAIR lncRNA. hnRNP A2 and B1 are splice isoforms of the same gene. The spliced version of HOTAIR preferentially associates with the B1 isoform, which we hypothesize contributes to RNA-RNA matching between HOTAIR and transcripts of target genes in breast cancer. Here we used enhanced cross-linking immunoprecipitation (eCLIP) to map the direct interactions between A2/B1 and RNA in breast cancer cells. Despite differing by only twelve amino acids, the A2 and B1 splice isoforms associate preferentially with distinct populations of RNA in vivo. Through cellular fractionation experiments we characterize the pattern of RNA association in chromatin, nucleoplasm, and cytoplasm. We find that a majority of interactions occur on chromatin, even those that do not contribute to co-transcriptional splicing. A2/B1 binding site locations on multiple RNAs hint at a contribution to the regulation and function of lncRNAs. Surprisingly, the strongest A2/B1 binding site occurs in a retained intron of HOTAIR, which interrupts an RNA-RNA interaction hotspot. In vitro eCLIP experiments highlight additional exonic B1 binding sites in HOTAIR which also surround the RNA-RNA interaction hotspot. Interestingly, a version of HOTAIR with the intron retained is still capable of making RNA-RNA interactions in vitro through the hotspot region. Our data further characterize the multiple functions of a repurposed splicing factor with isoform-biased interactions, and highlight that the majority of these functions occur on chromatin-associated RNA.

Keywords: HOTAIR; RNA-RNA interactions; Xist; chromatin; hnRNP A2/B1; long noncoding RNA.

Figure 1.

Conservation of the hnRNP B1-specific exon across species. (a) Model of protein domains of hnRNP A2 and B1, with B1-specific domain highlighted. (b) Multiple sequence analysis of hnRNP B1 genomic sequence in human, mouse, rat, cow, elephant, and opossum reference sequences, with B1-specific exon and next downstream exon highlighted, demonstrating high degree of conservation of the B1-specific exon in all eutherian species. Mismatches from human sequence are colored in red. (c) Immunoprecipitation using antibody specific to hnRNP B1 specifically retrieves hnRNP B1 in both human and mouse samples. (d) RT-PCR primers surrounding B1-specific exon 2 demonstrate inclusion of B1 exon in total RNA from both human MCF7 and mouse C2C12 cells.

Figure 2.

A2/B1 and B1 eCLIP results in MCF7 cells. (a) Schematic of the eCLIP protocol(b) Irreproducible Discovery Rate (IDR) analysis comparing peak fold enrichment indicates increased reproducibility for replicates compared against one another (A2/B1, black; B1, red) than comparison of replicates from A2/B1 and B1 experiments (purple).(c) Correlation of A2/B1 eCLIP replicate fold enrichment at non-significant (black) or significant (red) peaks in replicate 1. R values are Pearson’s correlation coefficient.(d) Correlation of B1 eCLIP replicate fold enrichment at non-significant (black) or significant (red) peaks in replicate 1. R values are Pearson’s correlation coefficient.(e) Correlation of A2/B1 and B1 eCLIP fold enrichment at non-significant (black) or significant (red) peaks in A2/B1. R values are Pearson’s correlation coefficient.(f) Example hnRNP A2/B1 eCLIP data, demonstrating region of the transcription factor Hivep3 that contains binding sites (black) enriched specifically in hnRNP B1 (blue). Y-axis scale is normalized to reads per million. Region pictured is chr1:42,345,550–42,344,700.(g) Analysis of RefSeq transcripts containing input normalized peaks in both replicates of A2/B1 or B1 eCLIP-seq experiments. Also pictured are analyses of transcripts that contain peaks only in introns or exons/UTRs.(h) Distribution of peaks (conserved between both replicates) in different areas of transcripts in hnRNP A2/B1 and B1 eCLIP experiments, compared to experiments performed in non-tumorigenic MCF10A cells. ‘Proximal introns’ are defined as intronic regions within 2 kb of an exon.(i) Top two identified motifs in both replicates of hnRNP A2/B1 and B1 eCLIPs in MCF7 and MCF10A cells.

Figure 3.

eCLIP of MCF7 chromatin, nucleoplasm, and cytoplasm. (a)Western blot of fractionated MCF7 cells (top) demonstrates preferential localization of A2/B1 to chromatin in fractions (either 1.5% or 5%) of an eCLIP fractionation experiment. Western blot of eCLIP samples (bottom) indicates preferential localization of A2/B1 to chromatin, as well as specific immunoprecipitation of B1 isoform.(b)The vast majority of A2/B1 binding peaks were identified in the chromatin sample, with a small minority overlapping with a nucleoplasm or cytoplasm binding peak. A small fraction of binding peaks were unique to either nucleoplasm or cytoplasm.(c) Compared to the chromatin sample, the nucleoplasm and cytoplasm peaks were far more likely to identify binding to either 5′ UTR or proximal intronic sequence.(d) The 5′ UTR of SYNCRIP is bound by B1 in both chromatin and nucleoplasm fractions. However, nearby intronic binding peaks are found only in chromatin fraction.(e) In the 3′ UTR of SEC14L1, there are two distinct binding peaks for B1. However, in nucleoplasm (purple) and cytoplasm (yellow), B1 preferentially binds between the two chromatin peaks.(f) A nucleoplasm-specific B1 binding peak in the small Cajal body-associated RNA SCARNA10.

Figure 4.

Binding of lncRNAs by hnRNP A2/B1. (a) hnRNP A2/B1 interacts with a small number of previously-identified lncRNAs.(b) hnRNP A2/B1 binding sites within the RepA region of Xist. RBM15 and RBM15b iCLIP tracks published in [42].(c) An hnRNP A2/B1 binding site within the lncRNA NORAD. PUM2 eCLIP replicate peaks retrieved from ENCODE Project experiment accession ENCSR661ICQ[15].(d) An hnRNP A2/B1 binding site within an intron of the lncRNA TUG1.(e) Native RNA immunoprecipitation demonstrating association of A2/B1 and selected lncRNAs.

Figure 5.

Investigation of a novel hnRNP A2/B1 binding site in the lncRNA HOTAIR. (a) Location of an hnRNP A2/B1 eCLIP peak in an intron of HOTAIR, downstream of the proposed PRC2 binding site (visible in the input library).(b) The intron between exons 3 and 4 of HOTAIR retained in a small fraction of HOTAIR MCF7 total RNA.(c) Intron 3 disrupts an RNA-RNA interaction site between HOTAIR and multiple gene target transcripts.(d) Graph depicting the total number of interactions (y-axis) between each nucleotide of the HOTAIR transcript and the top 1% of transcripts (440 RNAs) predicted to interact with HOTAIR[50]. The nucleotide position within the HOTAIR transcript (x-axis) has been shifted upstream (−139nt) so that it accurately corresponds to the TSS of the transcript variant GenBank accession DQ926657.1. The highest number of RNA-RNA interaction events is found between 226 to 297 nt (red box).(e) Schematic of in vitro eCLIP experiment using recombinant B1 protein and in vitro transcribed HOTAIR.(f) In vitro eCLIP of recombinant B1 and in vitro transcribed mature form of HOTAIR identified binding sites in exon 1 and in exons 5–6 of HOTAIR (exons alternately shaded blue), with minimal binding identified in non-crosslinked control. Comparison is made to exonic in vivo B1 eCLIP signal (red).(g) In vitro RNA-RNA interaction assays with RAT-tagged JAM2 and either full-length HOTAIR, HOTAIR with intron 3 or Anti-Luc RNA in the presence or absence of hnRNP B1. The association of HOTAIR and Anti-Luc RNA with JAM2 was quantified by RT-qPCR (n = 6).

Figure 6.

Model of hnRNP B1 interaction with HOTAIR. (a) hnRNP B1 on chromatin can bind to transcripts co-transcriptionally, or bind to lncRNAs associating with other loci in trans.(b) On the lncRNA HOTAIR, hnRNP B1 preferentially binds to intron 3, which bisects an RNA-RNA interaction hotspot. Retention of intron 3 may not disrupt the ability of HOTAIR to pair with other RNAs in the hotspot region. Following splicing of intron 3, B1 remains bound to nearby regions of HOTAIR, continuing to promote RNA-RNA interactions from the hotspot region.