Figure 5. Polyribosome association of endogenous SF2/ASF mRNA targets. A) Western blot analysis of cytoplasmic extracts prepared from cells transfected with pCGT7 of pCGT7 SF2/ASF (lanes 1 and 2, respectively). Blots were probed with anti-SF2/ASF and anti- GAPDH (left and right panel, respectively). B) Polyribosome association of endogenous mRNAs. Cytoplasmic extracts from control or pCGT7 SF2/ASF cells were resolved by sucrose gradient fractionation. The upper panel shows the absorbance of rRNA across the gradient, the SF2/ASF RNA Targets positions of ribosomal subunits and complexes are indicated. Lower panels: RT-PCR analysis of RNA targets. Products were resolved by agarose gel electrophoresis. The sub-cellular fraction corresponding to the initial CLIP experiment is indicated below each gene symbol. doi:10.1371/journal.pone.0003369.g005 Polyribosome Profiles Polyribosome profiles were obtained from HEK293T cells as previously described [7,8]. Total RNA was extracted from each fraction using TRI Reagent LS. Every other fraction was analyzed by RT-PCR. Purification of polyribosome for CLIP analysis of SF2/ ASF was accomplished by pelleting polyribosomes through 10–25% sucrose gradients as previously described [8]. Polysome pellets were washed gently with cold PBS, then resuspended in cold 0.56 buffer A (see above). The polysomes were then treated with RQ DNase, and RNase as described in the CLIP protocol [12]. Supporting Information A supplementary excel spread sheet accompanies this manuscript (Table S1). It contains complete annotation of the CLIP data. Supporting Information Table S1 A complete list of binding site annotation using the Ensembl, UCSC Known Gene and Rfam databases. The Excel file can be filtered in order to find binding sites identified by CLIP using nuclear, cytoplasmic or polysomal extracts. Headers for the table are as follows: Chromosome: Defines the specific chromosome from the human genome to which the seq-block mapped. Strand: CLIP preserves the orientation of the captured RNA marker, so that it is possible to determine the strandedness of the locus. Id: Generic description given to each binding site during our annotation work flow. Region start/end: The precise chromosomal coordinates defining the unique or overlapping sequence blocks. Regions are define by at least 2 partially overlapping binding sites mapping to the locus. # of fragments in the Cytoplasm/Nucleus/Polysome indicates if the binding site was identified in each compartment. The number specifies whether a sequence block was absent, present in a single assay or multiple assays. # of sample targets is useful for finding binding sites that are in 1, 2 or all three cellular fractions. Gene Annotation: This column describes the relationship of the seq block to annotated protein coding genes based on the UCSC Known Gene Database. Exon Style: This column describes the relationship of the seq block to annotated parts of protein coding genes (exon, intron etc). The strategy is presented in Supplementary Figure 2. USCS Known Gene database ID: This column refers to the name of a specific gene cluster by the UCSC Known Gene database. Gene Symbol: This column contains information pertaining to the approved HUGO Gene Nomenclature Committee symbol for each protein coding gene. Exon Position: This column describes the position of the exon within the protein coding gene. First/Last exon columns: Designation of ‘‘1’’ in either column indicates 59 or 39 terminal exon. ‘‘1’’ in both columns denotes that the sequence block is in a single exon gene. Upstream/Downstream Exon Position: These columns are useful for determining the position of introns within a protein coding gene. ncRNA Annotation: Describes the relationship of a sequence block to annotated non coding RNA (ncRNA). Annotation is based on the Rfam database. ncRNA Name: This column describes the gene symbol for each ncRNA containing a sequence block. UTR type: Describes the relationship between sequence blocks and untranslated regions of protein coding genes. Splicing Event: Gives alternative splicing annotation for exonic binding sites based upon AceVIEW, ALT Events and Fast-db, databases. Found at: doi:10.1371/journal.pone.0003369.s001 (0.13 MB XLS) PLoS ONE | www.plosone.org 9 October 2008 | Volume 3 | Issue 10 | e3369