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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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Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1a, Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a, presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex12,14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log2) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61.
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Figure 6: Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1a, Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a, presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex12,14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log2) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61.

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)

Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1a, Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a, presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex12,14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log2) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61.
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Figure 6: Autoradiography analysis of the STAU1-RNA complex, and analysis of hybrid reads in a known STAU1 mRNA target, ARF1a, Autoradiograph of STAU1-RNA complex that was isolated for the hiCLIP experiment. hiCLIP experiments were performed with high and low RNase conditions, and the two controls omitted either the 2nd intermolecular ligation or STAU1 induction. After adaptor ligation, STAU1 cross-linked RNA was radio labeled and the complex was analyzed by denaturing gel electrophoresis and membrane transfer. The size of the band is slightly higher compared to that in Fig. 1a, presumably due to the efficient adaptor ligation that adds to the size of the RNAs (the experiment shown in Fig. 1a didn’t include adaptor ligation). b, Correlation analysis of the non-hybrid read count on each RNA between the replicates of the hiCLIP experiments. c, Schematic representations of ARF1 mRNA and the known STAU1-target RNA duplex, along with the position of STAU1 hybrid reads and cross-link sites identified by non-hybrid reads. The left and right arms of hybrid reads are depicted as black boxes, and lines connect reads originating from the same cDNA. The previously studied STAU1-target RNA duplex12,14 is indicated by green and red boxes. In addition to the known duplex, hybrid reads also identified additional duplexes in the ARF1 3′ UTR. Interestingly, two newly identified duplexes are part of overlapping secondary structures, both of which represent the minimum free energy of folding the local sequence, as predicted by RNAfold24 (shown on the right). This suggests that some regions of the ARF1 3′ UTR may adopt alternative conformations. The overlapping region of the two structures is shaded in blue. d, The constructs of reporters (ARF1 WT and Δ) used for the validation by formaldehyde crosslinking and co-immunoprecipitation experiment are shown. The reporter has firefly luciferase (FLuc) CDS and ARF1 3′ UTR. e, The ratio of ARF1 WT and Δ in total cell lysate fraction or STAU1 co-immunopreciptated fraction were analyzed by RT-PCR using forward primer annealed to CDS of FLuc and reverse primer annealed to downstream of the deletion site. The ratios (log2) of two populations are compared by Welch’s t test (n = 3). The corresponding Qiaxcel electropherograms are available at: figshare.com/s/5f83e88e929b11e4b77106ec4b8d1f61.
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.

Show MeSH