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.

HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells

“…In MCF-7 ERα+ breast cancer cells, enhanced cross-linking immunoprecipitation (eCLIP) using antibodies specific to HNRNPB1 alone or HNRNPA2/B1 in combination identified 1,472 transcripts bound by both HNRNPB1 and HNRNPA2/B1, 899 transcripts uniquely bound by HNRNPB1, and 479 transcripts uniquely bound by HNRNPA2/B1 32 . HNRNPB1 binding sites revealed a preference for 5′-AGGAAGG-3′ versus 5′-UGGGGA-3′ for HNRNPA2/B1 32 . HNRNPA2/B1 binding peaks were primarily in chromatin samples, consistent with HNRNPA2/B1 binding to nascent transcripts 32 .…”

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

“…hnRNP A2/B1 has previously been suggested to be a reader of m6A, and the B1 isoform has a high affinity for binding HOTAIR 24,30,31 . However, comparing our previously generated eCLIP results for hnRNP B1 32 to the m6A eCLIP, both performed in MCF-7 cells, we found that, out of 10,470 m6A sites, only 417 (4%) were identified to contain an hnRNP B1 binding site within 1,000 nucleotides ( Figure S4A). Upon mapping hnRNP B1 signal intensity relative to the nearby m6A site, we observed that hnRNP B1 is depleted directly over m6A sites ( Figure S4B).…”

Knockdown of LINC00662 represses AK4 and attenuates radioresistance of oral squamous cell carcinoma

“…Past researches have unveiled that RNA-binding proteins (RBPs) can interact with lncRNAs in tumors [23]. From starBase, we discovered that LINC00662 potentially bound with hnRNPC protein (Fig.…”