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hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1.

Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D'Ambrogio A, Luscombe NM, Ule J - Nature (2015)

Bottom Line: Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions.We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability.Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

ABSTRACT
The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

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STAU1 regulates cytoplasmic splicing of XBP1 mRNAa, Schematic representation of the unspliced XBP1(u) and spliced XBP1(s) mRNA, shown together with hybrid reads that identify RNA duplexes.b, Proportion of XBP1(s) mRNA 30 min after ER stress induction in untreated (UT), STAU1 knockdown (KD), and rescued (RC) cells. The proportions of XBP1(s) in different conditions were compared using the two-tailed Welch’s t test (n = 9 from 3 independent experiments).c, Schematic diagram summarizing the functional analyses of STAU1-target mRNAs. 3′ UTR bound mRNAs tend to be highly translated and encode membrane proteins that are translated at the ER (in gray). CDS bound mRNAs tend to be lowly translated and encode proteins that function in the cell cycle M phase. Loops formed by RNA duplexes in the 3′ UTR tend to be longer than in the CDS.
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Figure 4: STAU1 regulates cytoplasmic splicing of XBP1 mRNAa, Schematic representation of the unspliced XBP1(u) and spliced XBP1(s) mRNA, shown together with hybrid reads that identify RNA duplexes.b, Proportion of XBP1(s) mRNA 30 min after ER stress induction in untreated (UT), STAU1 knockdown (KD), and rescued (RC) cells. The proportions of XBP1(s) in different conditions were compared using the two-tailed Welch’s t test (n = 9 from 3 independent experiments).c, Schematic diagram summarizing the functional analyses of STAU1-target mRNAs. 3′ UTR bound mRNAs tend to be highly translated and encode membrane proteins that are translated at the ER (in gray). CDS bound mRNAs tend to be lowly translated and encode proteins that function in the cell cycle M phase. Loops formed by RNA duplexes in the 3′ UTR tend to be longer than in the CDS.

Mentions: The mRNA encoding XBP1 (also known as endoplasmic reticulum to nucleus signaling 1; ERN1), a central player in ER stress response27, was one of the mRNAs with substantially increased abundance upon STAU1 knockdown (Extended Data Fig. 7f). hiCLIP identified a duplex spanning 858nts in the XBP1 3′ UTR, which is required for efficient splicing of the XBP1 transcript during ER stress (Fig. 4a, b, Extended Data Fig. 8a-d). We produced three reporter constructs to examine the effect of this long-range duplex on the stability of the XBP1 transcript; one containing the XBP1 3′ UTR with the original sequence, one with an AA dinucleotide insertion to disrupt the duplex, and one with an additional insertion of a complementary TT dinucleotide to restore the duplex structure (Extended Data Fig. 8a). We observed that the AA insertion decreased mRNA stability, whereas the complementary TT mutation restored it back to original levels (Extended Data Fig. 8e).


hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1.

Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D'Ambrogio A, Luscombe NM, Ule J - Nature (2015)

STAU1 regulates cytoplasmic splicing of XBP1 mRNAa, Schematic representation of the unspliced XBP1(u) and spliced XBP1(s) mRNA, shown together with hybrid reads that identify RNA duplexes.b, Proportion of XBP1(s) mRNA 30 min after ER stress induction in untreated (UT), STAU1 knockdown (KD), and rescued (RC) cells. The proportions of XBP1(s) in different conditions were compared using the two-tailed Welch’s t test (n = 9 from 3 independent experiments).c, Schematic diagram summarizing the functional analyses of STAU1-target mRNAs. 3′ UTR bound mRNAs tend to be highly translated and encode membrane proteins that are translated at the ER (in gray). CDS bound mRNAs tend to be lowly translated and encode proteins that function in the cell cycle M phase. Loops formed by RNA duplexes in the 3′ UTR tend to be longer than in the CDS.
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Figure 4: STAU1 regulates cytoplasmic splicing of XBP1 mRNAa, Schematic representation of the unspliced XBP1(u) and spliced XBP1(s) mRNA, shown together with hybrid reads that identify RNA duplexes.b, Proportion of XBP1(s) mRNA 30 min after ER stress induction in untreated (UT), STAU1 knockdown (KD), and rescued (RC) cells. The proportions of XBP1(s) in different conditions were compared using the two-tailed Welch’s t test (n = 9 from 3 independent experiments).c, Schematic diagram summarizing the functional analyses of STAU1-target mRNAs. 3′ UTR bound mRNAs tend to be highly translated and encode membrane proteins that are translated at the ER (in gray). CDS bound mRNAs tend to be lowly translated and encode proteins that function in the cell cycle M phase. Loops formed by RNA duplexes in the 3′ UTR tend to be longer than in the CDS.
Mentions: The mRNA encoding XBP1 (also known as endoplasmic reticulum to nucleus signaling 1; ERN1), a central player in ER stress response27, was one of the mRNAs with substantially increased abundance upon STAU1 knockdown (Extended Data Fig. 7f). hiCLIP identified a duplex spanning 858nts in the XBP1 3′ UTR, which is required for efficient splicing of the XBP1 transcript during ER stress (Fig. 4a, b, Extended Data Fig. 8a-d). We produced three reporter constructs to examine the effect of this long-range duplex on the stability of the XBP1 transcript; one containing the XBP1 3′ UTR with the original sequence, one with an AA dinucleotide insertion to disrupt the duplex, and one with an additional insertion of a complementary TT dinucleotide to restore the duplex structure (Extended Data Fig. 8a). We observed that the AA insertion decreased mRNA stability, whereas the complementary TT mutation restored it back to original levels (Extended Data Fig. 8e).

Bottom Line: Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions.We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability.Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

ABSTRACT
The structure of messenger RNA is important for post-transcriptional regulation, mainly because it affects binding of trans-acting factors. However, little is known about the in vivo structure of full-length mRNAs. Here we present hiCLIP, a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs). Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells, we uncover a dominance of intra-molecular RNA duplexes, a depletion of duplexes from coding regions of highly translated mRNAs, an unexpected prevalence of long-range duplexes in 3' untranslated regions (UTRs), and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions. We also discover a duplex spanning 858 nucleotides in the 3' UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability. Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new, especially long-range, RNA duplexes.

Show MeSH