Urb-RIP enriches for mCherry-mRNA and bound PABP.

<p><b>a</b> Schematic describing 2HA-Urb construct used for Urb-RIP and reporter, mCherry (mCh)-mRNA tagged with SLII, used to validate and optimize Urb-RIP. <b>b</b> Enrichment of mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with a plasmid expressing mCherry-mRNA untagged or tagged with SLII. Two days after transfection the cells were UV-irradiated at 400 mJ/cm² and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh and GAPDH primers. <b>c</b> Comparison of relative expression amounts of mCherry with (+SLII) and without (-SLII) tagging. qPCR results show a modest reduction in mCherry expression upon insertion of SLII. Relative levels of mCherry are normalized to GAPDH. <b>d</b> western blot shows enrichment of PABP following Urb-RIP of mCh. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. <b>e</b> quantification of western blot in <b>d</b>. For panels <b>b</b> and <b>c</b> mean ± SD of three independent experiments are shown. For panel <b>e</b> mean ±SD of three independent experiments are shown. * p<0.05, ** p<0.01.</p

Schematic illustration of the Urb-RIP protocol and potential applications.

<p>The first step of the Urb-RIP protocol is to tag an RNA of interest with the SLII-tag, illustrated in the figure. The tagged RNA and in parallel an untagged control are coexpressed with 2HA-Urb in a cell line of interest. After a period of time the cells are UV irradiated to produce RNA-protein crosslinks. The cells are then lysed and immunoprecipitation is performed using blocked anti-HA magnetic beads. The RNA or protein is then eluted and analyzed by an appropriate method. This approach should be applicable to mRNAs and lncRNAs and amendable to a wide range of methods for eluate analysis.</p

Urb-RIP shows Argonaute and miRNA binding to miRNA-targeted messages in human cells.

<p><b>a</b> Schematic describing the <i>let-7</i> reporters used to validate Urb-RIPs ability to identify interacting miRNA. <b>b</b> Enrichment of <i>let-7</i> and mCh-mRNA by Urb-RIP as determined by qPCR. The cell line 293-2HA-Urb was transfected with plasmids expressing the constructs described in <b>a</b> as well as a plasmid for expression of GFP-FLAG-Ago2. Two days after transfection the cells were UV-irradiated at 400 mJ/cm² and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using mCh, <i>let-7</i> and GAPDH primers. <b>c</b> enrichment of <i>let-7</i> normalized to mCh abundance in the immunoprecipitate. <b>d</b> western blot shows enrichment of GFP-FLAG-Ago2 following Urb-RIP. The mCh-<i>let-7</i>-SLII reporters from above were co-transfected with GFP-FLAG-Ago2. Two days after transfection Urb-RIP was performed. The eluted protein as well as input was analyzed by western blot with antibody against FLAG (GFP-FLAG-Ago2 and 2HA-FLAG-Urb). Samples labeled input represent 5% of the total sample used for Urb-RIP. <b>e</b> quantification of western blot in <b>d,</b> normalized to 2HA-Urb, relative to mCh-<i>let-7</i>-SLII.</p

Urb-RIP confirms binding of PABP to BC200.

<p><b>a</b> Schematic describing the BC200 construct used for pull-down by Urb-RIP. <b>b</b> Enrichment of BC200+SLII by Urb-RIP as determined by qPCR. A stable HEK-293 cell line for the inducible expression of 2HA-Urb was transfected with plasmids expressing the constructs described in <b>a</b> Two days after transfection the cells were UV-irradiated at 400 mJ/cm² and subsequently lysed. Immunoprecipitation was performed using the Urb-RIP protocol and RNA was eluted with proteinase K treatment. qRT-PCR was performed using BC200 and GAPDH primers. <b>c</b> Comparison of relative expression amounts of BC200 with (+SLII) and without (-SLII) tagging. qPCR results show no change in BC200 expression upon insertion of SLII. Relative levels of BC200 are normalized to GAPDH. <b>d</b> Western blot shows enrichment of PABP following Urb-RIP of BC200+SLII. Half of the immunoprecipitate from above was eluted with sample buffer and analyzed by western blot with antibody against proteins listed. Samples labeled input represent 5% of the total sample used for Urb-RIP. <b>e</b> Analysis of western blot in <b>d</b>, normalized to 2HA-Urb, relative to mCh-SLII. For panels <b>b</b> and <b>c</b> mean ± SD of two independent experiments are shown. For panel <b>e</b> mean ±SD of three independent experiments are shown. * p<0.05.</p