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. 2008:488:85-98.
doi: 10.1007/978-1-60327-475-3_6.

CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins

Kirk B Jensen  1 , Robert B Darnell
Affiliations

CLIP: crosslinking and immunoprecipitation of in vivo RNA targets of RNA-binding proteins

Kirk B Jensen et al. Methods Mol Biol. 2008.

Abstract

We present a newly developed method for fixing RNA-protein complexes in situ in living cells and the subsequent purification of the RNA targets. Using this approach, complex tissue such as mouse brain can be ultraviolet (UV) irradiated to covalently crosslink RNA-protein complexes. Once covalently bound, RNA-protein complexes can be purified under stringent conditions, allowing a highly specific purification scheme to be employed. After UV irradiation, the tissue is solubilized and the RNA partially digested, allowing a small fragment to remain attached to protein. RNA-protein complexes of interest are partially purified by immunoprecipitation and noncovalently associated RNA removed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These purified RNA-protein complexes are isolated and treated with proteinase K, which removes protein but leaves intact RNA. This RNA is abundant enough, and competent for, RNA linker ligation, reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and sequencing. Database matching of these short 70- to 100-nt RNA CLIP (crosslinking and immunoprecipitation of RNA-protein complexes) "tags," which mark the native binding sites of RNA binding proteins, potentially allows the entire target repertoire of an RNA binding protein to be determined.

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Figures

Fig. 1
Fig. 1
Autoradiograph of 32P-labeled RNA crosslinked to the neuronal Hu proteins. P4 mouse brain tissue was irradiated with short-wavelength ultraviolet (UV) light. The tissue was collected, and the soluble extracts were treated with ribonuclease (RNase) T1 and immunoprecipitated with antiserum specific for the neuronal Hu proteins (HuB, HuC, and HuD). The purified material was labeled with 32P and run on reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel contents were transferred to nitrocellulose by wet transfer; the filter was blotted dry and exposed to film. The lane with reducing agent shows the interference caused by the approximately 50-kDa heavy chain “pushing” the gel contents out of its way. In the nonreducing lane, a fraction of the membrane from approximately 40 to 60 kDa was used for purification of the RNA–protein complexes. (The Hu proteins usually run at about 35 kDa.)
Fig. 2
Fig. 2
Gel purification of the CLIP (crosslinking and immunoprecipitation of RNA– protein complexes) RNA after linker ligation. RNA before and after linker ligation was run on a 7 M urea/20% polyacrylamide gel. The modal size of this RNA pool before ligation was approximately 30 nt. After ligation, the modal size of the RNA increased to about 50 nt. The postligation pool is probably composed of significant fractions of single-linker ligation (CLIP RNA of 30 nt plus one linker of 16 nt) and smaller amounts of both 5′ and 3′ linker ligations (CLIP RNA of 30 nt and linkers of 35 nt). Two fractions of RNA were isolated from this gel for reverse transcriptase polymerase chain reaction (RT-PCR). Fraction 1 is from approx 60 to approx 85 nt (with CLIP tag inserts of 25–50 nt), and fraction 2 is from approx 85 to approx 175 nt (with CLIP tag inserts of 50–140 nt).
Fig. 3
Fig. 3
Reverse transcriptase polymerase chain reaction (RT-PCR) of purified linker-ligated CLIP (crosslinking and immunoprecipitation of RNA–protein complexes) RNA. Fractions 1 and 2 of CLIP RNA gel purified after linker ligation (Fig. 2) were used to make complementary DNA for polymerase chain reaction (PCR) amplification. The PCR products obtained from fraction 1 (lane 1) and fraction 2 (lane 2) were run on a 7 M urea/20% polyacrylamide gel. Fraction 1 PCR products primarily cover a range from 50 to 80 nt, while those from fraction 2 fall within a range of 70 to 125 nt. In general, these sizes are consistent with the sizes of RNA taken from the gel purification of the linker-ligated CLIP RNAs (both fractions of PCR products are slightly shorter than predicted). To obtain CLIP tag inserts of 30 nt or greater, only PCR products great than 65 nt should be gel purified and used for the final PCR reaction before cloning. Also note the prominent product at about 38 nt. We believe that this is a PCR product resulting from amplification of a linker-linker ligation (a very prominent side reaction of the linker ligation step). While this RNA should be removed during gel purification of the RNA, its abundance makes it very difficult to get rid of it. After gel purification of the correct PCR products, this band will disappear (thus, it is not a PCR-generated “primer-dimer”).
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