CMV Intron SV 40 pA F PE G RNA probe FLAG ORF1 ORF2 CMV AATAAA 5’UTR 3’UTR BGH (p)AL1(p)A FL-O1F A % EGFP positive cells 1. pCDNA ± FL 2.55 ± FL-O1F 2.30 ± Transfect L1 constructs into HEK 293T cells 72 hours FACS analysis Constructs B Figure S1 Transfect L1 constructs into HEK293T cells Cytoplasmic lysate incubate with anti-FLAG beads for one hour Beads washed and the RNPs were eluted with FLAG peptide competition C hours Fig S1 L1 retrotransposition assay of engineered L1: A) An EGFP retrotransposition indicator cassette is cloned in the Ale I restriction site in the 3’-UTR of L1 to check the retrotrotransposition efficiency of an engineered L1 (60). B) Marked L1 was transfected into HEK293T cells and retrotransposition was measured 72 hours post transfection. Retrotransposition activity was scored as the number of EGFP positive cells/number of transfected cells (%EGFP) ± standard error of the mean. Wild type L1RP (FL) and empty vector (pcDNA6-FLAG) served as positive and negative controls, respectively. C) Flow chart of L1RNP purification.

Transfected plasmid construct in 50% confluent 293T cells 4-SU added after 24 hours and cells grown for another 12 hours Cells irradiated at 365nm UV on culture plates Lysed, treated with RNAseT1 and immunoprecipitated with anti-FLAG agarose beads Washed beads, treated again with RNAse T1 before labeling RNA with γ32P-ATP Boiled beads with SDS-PAGE gel loading buffer and separated labeled RNA –protein complex in SDS –PAGE gel Exposed radioactive gel to X-ray film Aligned radioactive gel to X-ray film to excise radioactive RNA-protein complex Separated RNA-protein complex from excised PAGE gel slice by electro elution and digested with Proteinase K Prepared cDNA library using small RNA cloning protocol ( Illumina) Deeply sequenced on Illumina platform A Figure S2 Fig S2 Flow-chart of PAR-CLIP technique.

Figure S3 Alu Total C 294 nt Alu Del Linker ORF1p (40 kDa) Western Agarose gel 1 2 D RNA marker 500 nt nt (AluT) 154 nt (Alu R) 114 nt (Alu L) Alu Left Alu Right ORF1p (40 kDa) Denatured PAGE Western 1 2 FL-O1F ORF1F HuR1F A Alu L Alu R AAAA AAAAn B Transfect ed ORF1F Construct 48 hours Cytoplasmic lysate incubated with bio- tinylated Alu RNA for 30 min at RT Beads boiled with 1X SDS-PAGE gel loading dye and resolved in SDS-PAGE gel ORF1p detected with FLAG Ab Streptavidin beads Washed with high salt FLAG ORF1

Fig S3 In vitro Alu RNA-ORF1p binding: A) Aligning sequence reads with Alu RNA. RNA reads with T to C mismatches on the forward strand and A to G mismatches on the reverse strand align with Alu sequence. The number of reads is on the Y axis and the sequence position in Alu RNA is on the X axis. Compared to HuR, both ORF1F and FL-O1F show significant binding in the A rich linker region (marked with a red line) and the poly A sequence (red line) present at the end of Alu. Schematic representation of an Alu RNA where linker sequence and poly A sequence marked in red. B) Flow-chart of Alu RNA and ORF1p binding assay. Cytoplasmic lysate was prepared after transfecting L1-ORF1F construct. Biotin labeled Alu RNA was incubated with lysate for 30 minutes at 25°C. The RNA-protein complex was purified with streptavidin-coupled dynabeads. The complex was resolved in a SDS-PAGE gel and ORF1p was detected by an immunoblot using an anti-FLAG antibody. C) Panel 1- Western blot detection of ORF1p with an anti-FLAG antibody showed that deletion of linker sequence severely reduced ORF1 binding with full length Alu RNA. Panel 2- Biotin labeled Alu RNA used for the assay was checked on a 2.0 % agarose gel. D) Panel 1-ORF1p binding assay with Alu left (AluL) or Alu right (AluR) monomer alone showed severe reduction compared to full length Alu RNA (AluT). Panel 2- Biotin labeled RNAs were checked on a 5% denatured PAGE gel. AluW- Alu wild; Alu Del L-Linker deleted Alu sequence.

Figure S4 Fig S4 Distribution of mapped reads with T>C (plus strand) changes relative to exon annotations. The height of the bars represent the fraction of total mapped reads that correspond to each exon category.

Figure S5 Fig S5 Fraction of mapped reads with T>C (plus strand) changes mapping to processed pseudogene annotations in FL-O1F, ORF1F, and HuR.

Figure S6

Fig S6 Comparison of HuR binding profiles from Kishore et al. (59) to samples generated in this study. All values represent Spearman correlation coefficients (  ) between binned normalized read counts. (a) Neighbor-joining tree based on hierarchical clustering of correlation coefficients shown in (b), a histogram and guide to the correlation values is shown under the plot. The correlation coefficients in the orange box indicated in (b) are shown as a bar chart in part (c). We obtained data generated in Kishore et al (59) for HuR PAR-CLIP treated with complete T1 digestion from GEO (accession GSE28859, samples GSM and GSM714638). These data were generated from 293T cells using a protocol that differs slightly from our own. Reads from these samples were trimmed for adapter sequences, aligned to the reference genome, and selected for potential PAR-CLIP-induced mutations using the same methods as were used on our PAR-CLIP samples. We then divided the genome into 10kb bins and counted the number of reads in each sample for each bin, and normalized to the total number of mapped reads in each sample, yielding a vector of binned normalized read counts for each sample. Comparing these vectors to one another, we see the Spearman correlation between Kishore et al. HuR samples and our samples is highest for our HuR sample [Figure S5(a &c)], demonstrating that the PAR-CLIP binding profile from the Kishore et al. HuR samples most closely resembles the HuR sample out of the samples in our study, although likely due to methological differences between Kishore et al. and the present study, we see that our samples overall more closely resemble one another (Figure S6b).

Figure S7 Fig S7 RPLP1, GAPDH, β-actin are reduced in ORF2(EN - )RNPs compared to FL-O1F RNPs. Equal amounts of cell lysate were used to purify RNPs from FL-O1F, ORF2(EN - ) and untransfected control samples. Isolated RNA was converted to cDNA and used as template to amplify RPLP1, GAPDH, and β-actin using SYBR Green PCR master mix (Qiagen) in ViiA™ 7 Real-Time PCR System (Applied Biosystems). ΔΔCt values were plotted to obtain fold enrichment (normalized to values obtained from FL-O1F). qRT-PCR analysis showed RPLP1, GAPDH and β-actin transcripts in ORF2(EN - )RNPs were 9, 5, and 3 %, respectively, of their levels in FL-O1F RNPs. Untransfected control showed no enrichment for any transcripts. ViiA™ 7 Real-Time PCR System RPLP1 GAPDH Actin ORF2ENmF FL-O1F Untransfect