CMV

Intron

SV 40 pA F P E G

RNA probeFLAG

ORF1 ORF2

CMV

AATAAA AATAAA5’UTR 3’UTR BGH (p)A L1(p)A

FL-O1F

A

% EGFP positive cells1. pCDNA6 0.01 ± 0.0022. FL 2.55 ± 0.0013. FL-O1F 2.30 ± 0.004

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

36-48 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

Tota

l

C

Alu

Tota

l

294 nt

Alu

Del

Lin

ker

ORF1p (40 kDa)

Western

Agarose gel

1

2

D

RNA marker

500 nt

300

200

100

294 nt (AluT)

154 nt (Alu R) 114 nt (Alu L)

Alu

Left

Alu

Righ

t

ORF1p

(40 kDa)

Denatured PAGE

Western

1

2

FL-O1F

ORF1F

HuR1F

A

Alu L Alu RAAAA 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 GSM714637 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.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1 2 3 4

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1 2 3 4

0.000.200.400.600.801.001.201.40

1 2 3 4

RPLP1

GAPDH

Actin

ORF

2EN

mF

FL-O

1F

Unt

rans

fect