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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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hiCLIP identifies RNA duplexes bound by STAU1a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction.b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a) was omitted.c, Mapping summary of the arms of hybrid reads.d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both).e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA (Extended Data Fig. 4b) are marked in a different colour for each region.f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).
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Figure 1: hiCLIP identifies RNA duplexes bound by STAU1a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction.b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a) was omitted.c, Mapping summary of the arms of hybrid reads.d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both).e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA (Extended Data Fig. 4b) are marked in a different colour for each region.f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).

Mentions: We performed hiCLIP from cytoplasmic extracts of Flp-In T-REx 293 cells. To recover a broad spectrum of RNAs and to ensure that only directly bound duplexes are identified, we employed two different RNase concentrations and stringent purification conditions (Fig. 1a, Extended Data Fig. 2a). We obtained 35,358 hybrid reads whose arms could be mapped to non-contiguous segments of RNA transcripts (Extended Data Fig. 1a-c, Supplementary table 1). Hybrid reads comprised approximately 2% of all reads. The remaining non-hybrid reads (1.2 million reads including control library; Supplementary table 1) were equivalent to traditional iCLIP reads and defined STAU1 cross-link sites19 (Extended Data Fig. 1a, Step 6). In contrast, hybrid reads comprised just 0.06% of control experiments omitting the second ligation reaction (Fig. 1b). Despite different RNase concentrations between replicates, there was good correlation in the numbers of reads mapping to each mRNA transcript (r=0.876; Extended Data Fig. 2b).


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)

hiCLIP identifies RNA duplexes bound by STAU1a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction.b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a) was omitted.c, Mapping summary of the arms of hybrid reads.d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both).e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA (Extended Data Fig. 4b) are marked in a different colour for each region.f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).
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Figure 1: hiCLIP identifies RNA duplexes bound by STAU1a, Autoradiography analysis of the STAU1-RNA complex at different RNase I concentrations or in the absence of cross-linking or STAU1 induction.b, The proportion of uniquely annotated hybrid reads in the hiCLIP libraries at high and low RNase conditions and from the control in which the second ligation (step 5 in Extended Data Fig. 1a) was omitted.c, Mapping summary of the arms of hybrid reads.d, Probability density distributions of minimum free energies of hybridization between the two arms of hybrid reads from mRNAs and long non-coding RNAs, or randomly repositioned sequences. Distributions were compared using the Mann-Whitney U test (n = 6120 for both).e, Alignment of three newly identified duplexes (hA, hB and hC) that connect distal regions of the human 18S rRNA. The nucleotide position and the nearest annotated helix from the CryoEM structure of the rRNA (Extended Data Fig. 4b) are marked in a different colour for each region.f, (Top) Proportion of hybrid reads that map to same or different RNA species. (Bottom) For hybrid reads mapping to same mRNA species, proportion in CDS, 3′ UTR, or other (i.e., 5′ UTR or spanning across two regions).
Mentions: We performed hiCLIP from cytoplasmic extracts of Flp-In T-REx 293 cells. To recover a broad spectrum of RNAs and to ensure that only directly bound duplexes are identified, we employed two different RNase concentrations and stringent purification conditions (Fig. 1a, Extended Data Fig. 2a). We obtained 35,358 hybrid reads whose arms could be mapped to non-contiguous segments of RNA transcripts (Extended Data Fig. 1a-c, Supplementary table 1). Hybrid reads comprised approximately 2% of all reads. The remaining non-hybrid reads (1.2 million reads including control library; Supplementary table 1) were equivalent to traditional iCLIP reads and defined STAU1 cross-link sites19 (Extended Data Fig. 1a, Step 6). In contrast, hybrid reads comprised just 0.06% of control experiments omitting the second ligation reaction (Fig. 1b). Despite different RNase concentrations between replicates, there was good correlation in the numbers of reads mapping to each mRNA transcript (r=0.876; Extended Data Fig. 2b).

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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Related in: MedlinePlus