The unspliced/spliced transcript ratio is miRNA-specific in B-cell lymphoma

For exonic miRNAs, such as miR-155, miR-22 and miR-146a, both unspliced and spliced transcripts include the complete stem-loop pre-miRNA sequence and may serve as the primary miRNA transcript. To discriminate between unspliced and spliced transcripts we designed qRT-PCR primer sets specific for unspliced or spliced transcripts as indicated in Figure 1A. We compared the levels of endogenous unspliced and spliced transcripts in twenty B-cell lymphoma cell lines with variable miRNA levels.

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Figure 1. The unspliced/spliced transcript ratio is miRNA-specific in B-cell lymphoma.

(A) Schematic overview of the unspliced and spliced BIC, pri-miR-22 and pri-miR-146a transcripts and location of the qRT-PCR products specific for unspliced, spliced and total (only for BIC) transcripts. Constructs used for miR-155 overexpression are also denoted. Short constructs, s-exBIC and s-intBIC, contain the pre-miR-155 sequence and ~150nt 3’ flanking sequence from exon 3 and 5’ flanking sequence derived from exon 2 (s-exBIC) or intron 2 (s-intBIC) of BIC transcript. The fl-exBIC construct covers the full-length spliced BIC transcript. Figures are not drawn to scale. (B) The median endogenous level of unspliced BIC transcript (0.049) was 3.7 fold higher than level of spliced BIC transcript (0.013). (C) The median endogenous level of unspliced pri-miR-22 (0.004) was similar to level of spliced pri-miR-22 (0.006). (D) The median endogenous unspliced pri-miR-146a level (0.004) was 20.5 fold lower than that of spliced pri-miR-146a (0.081). Levels of BIC, pri-miR-22 and pri-miR-146a were assessed in 20 B-cell lymphoma cell lines and normalized to HPRT levels. Significance was tested with Mann-Whitney test. (E) Both spliced and unspliced BIC showed a similar significant correlation with miR-155 levels. (F) Neither of the two pri-miR-22 transcripts showed a significant correlation with the miR-22 levels. (G) Both unspliced and spliced pri-miR-146a levels significantly correlated with the miR-146a levels. However, the slope of the regression line was significantly steeper for spliced pri-miR-146a.

https://doi.org/10.1371/journal.pone.0076647.g001

Endogenous miR-155 levels were highly variable in B-cell lymphoma cell lines. The difference between the cell line with the lowest (ST486) and the highest (OCI-Ly3) miR-155 level was ~500 fold (Figure S1A). The median level of the unspliced BIC transcripts was 3.7 fold higher than that of the spliced BIC transcripts (Figure 1B). The levels of endogenous unspliced BIC transcripts were higher than the levels of spliced BIC transcripts in 17 out of 20 cell lines irrespective of the miR-155 levels (Figure S1B).

MiR-22 levels were low in all B-cell lymphoma cell lines (Figure S1C). Pri-miR-22 has four alternative splice variants. Transcript variant 3 was almost exclusively detected in our panel of B-cell lymphoma cell lines (data not shown). We therefore restricted our subsequent analysis to this splice variant. The medians of endogenous levels of unspliced and spliced pri-miR-22 were similar in 20 analyzed cell lines (Figure 1C). The spliced/unspliced pri-miR-22 transcript ratio varied from 0.2 to 8 (Figure S1D). In SU-DHL-4 cells, the unspliced pri-miR-22 transcript was hardly detected.

MiR-146a levels varied over a 1000-fold range between B-cell lymphoma cell lines (Figure S1E). The levels of spliced pri-miR-146a transcripts were significantly higher than unspliced pri-miR-146a transcripts with a more than 20 fold difference in medians (Figure 1D). The spliced/unspliced pri-miR-146a transcript ratio varied from 14 to 72 fold (Figure S1F). In L540 cells only the spliced pri-miR-146a transcript was present.

Next, we correlated the mature miRNA levels with either unspliced or spliced pri-miRNAs levels. For miR-155, both unspliced and spliced BIC transcript levels showed significant correlation with miR-155 levels (R² = 0.53). The difference observed in levels of unspliced and spliced BIC transcripts did not result in a significant difference in the slope of the regression lines. For miR-22, no significant correlation was observed for either spliced or unspliced pri-miR-22 transcript levels with mature miR-22 levels. This might be caused by differences in the efficiency of the pri-miR-22 transcript to miR-22 processing, differences in the miR-22 stability, factors regulating the levels of mature miR-22 or limiting amounts of factors involved in the biogenesis of miRNAs. For miR-146a, both unspliced and spliced pri-miR-146a transcript levels significantly correlated with miR-146a (R² = 0.67 and 0.7, respectively). However, the slopes of the curves differed significantly (p<0.0001) due to lower unspliced pri-miR-146a transcript levels.

Thus, for all three exonic miRNAs, both unspliced and spliced primary transcripts are present, albeit at a variable ratio. For BIC, the unspliced primary transcript is predominant, for pri-miR-146a the spliced primary transcripts is predominant, whereas for pri-miR-22 the unspliced/spliced transcript ratio varies between cell lines. Levels of the mature miRNAs correlated with levels of both unspliced and spliced pri-miRNA transcripts for miR-155 and miR-146a, but not for miR-22.

Spliced pri-miRNA transcripts are partly transported to the cytoplasm

To further examine, whether unspliced or spliced pri-miRNA transcripts are available for miRNA processing, we examined the subcellular localization of unspliced and spliced transcripts of BIC, pri-miR-22 and pri-miR-146a by qRT-PCR. We determined the amount of the spliced and unspliced transcripts in the cytoplasm and the nucleus relative to their levels in the total fraction (Figure 2). To asses subcellular localization of unspliced and spliced BIC transcripts, we selected cell lines with low (L428), intermediate (Jiyoye) or high (L540) miR-155 levels (Figure S1A). The same cell lines were used for subcellular localization of pri-miR-22 and pri-miR-146a to allow comparison between subcellular distributions of pri-miRNA transcripts. We confirmed purity of the nuclear and cytoplasmic fractions by analyzing relative abundance of tRNA-Lys in the cytoplasm, and U3 and Xist in the nucleus (Figure S2A-C).

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Figure 2. Cellular localization of unspliced and spliced pri-miRNA transcripts.

(A) Unspliced BIC transcript levels were significantly higher in the nucleus than in the cytoplasm, whereas spliced BIC transcripts showed similar levels. (B) Unspliced pri-miR-22 transcripts were more abundant in the nucleus than in the cytoplasm and spliced pri-miR-22 transcript levels were similar. (C) Unspliced pri-miR-146a transcripts were almost exclusively in the nucleus, and spliced pri-miR-146a transcripts were both in the nucleus and in the cytoplasm. Unspliced and spliced pri-miR-146a transcripts were not detectable in L540 cells. Levels of pri-miRNA transcripts in the nucleus or the cytoplasm were calculated relative to their levels in the total fraction and corrected for the amount of RNA per cell. Average of three independent experiments was presented. Significance was calculated using 2-way ANOVA and Bonferroni posttest (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns - not significant).

https://doi.org/10.1371/journal.pone.0076647.g002

Endogenous unspliced BIC transcripts were located exclusively in the nuclear fraction in all three cell lines (Figure 2A). Spliced BIC transcripts showed similar amounts in the nucleus and in the cytoplasm. Localization of unspliced pri-miR-22 transcripts was predominantly, but not exclusively, nuclear in all three cell lines (Figure 2B). Spliced pri-miR-22 transcripts were present at similar levels in the nucleus and the cytoplasm in all three cell lines. Localization of unspliced pri-miR-146a transcripts was exclusively nuclear for both L428 and Jiyoye cells (Figure 2C). In L540 cells, both mature and primary miR-146a transcripts were hardly detected, so it was not possible to determine the subcellular localization. Spliced pri-miR-146a transcripts were more abundant in the nucleus than in the cytoplasm of L428 cells and in similar amounts in Jiyoye cells.

Thus, part of the spliced BIC, pri-miR-22 and pri-miR-146a transcripts are transported to the cytoplasm and as such unavailable for processing. Unspliced transcripts show an almost exclusive nuclear localization for BIC, pri-miR-22 and pri-miR-146a. Since the levels of spliced pri-miR-22 in L540 and Jiyoye cells and pri-miR-146a in Jiyoye and L428 were higher than the unspliced transcript levels (Figures S1D and S1F), spliced pri-miR-22 and pri-miR-146a may still be the predominant miRNA substrate.

Exogenous spliced BIC can be processed to miR-155

For further analysis we focused on miR-155, because previous studies have shown conflicting data concerning BIC to miR-155 processing [25,26]. The upstream pre-miR-155 flanking sequence is different in spliced and unspliced BIC transcripts. To determine if this upstream sequence affects the processing efficiency, we assessed the levels of miR-155 induction upon overexpression of BIC from two short fragments of BIC containing the stem-loop region, ~150nt 3’ flanking sequence from exon 3 and ~150nt 5’ flanking sequence derived either from intron 2 (s-intBIC) or from exon 2 (s-exBIC) of BIC transcript (Figure 1A). In addition, we also overexpressed the full-length spliced BIC (fl-exBIC) transcript (Figure 1A). We transduced these three BIC constructs into ST486, Ramos and U-HO1, i.e. cells that all have low endogenous miR-155 levels. A high expression of BIC was induced using either of the constructs (Figure 3A), albeit at variable levels. Transduction with the short exon spanning BIC construct resulted in the highest increase and the full-length spliced BIC construct resulted in the lowest increase in total BIC transcript levels (Figure 3A). Interestingly, the level of miR-155 induction was similar for all three constructs (Figure 3B), despite the marked differences in total BIC levels. These data indicate that the upstream pre-miR-155 flanking sequence does not modify processing efficiency and suggest that at a certain level of primary miRNA transcript, other factors become limiting or regulate the level of mature miR-155.

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Figure 3. Processing and cellular localization of exogenous BIC transcripts.

Levels of total BIC (A) and miR-155 (B) determined upon overexpression of the three constructs and empty vector (EV) in ST486, Ramos and U-HO1 cells. For the three BIC overexpression constructs, the increase in total BIC was variable and the highest levels were observed for the short BIC transcript containing the exon 2-derived 5’ pre-miR-155 flanking sequence (s-exBIC). However, miR-155 induction was similar for all BIC overexpression constructs. (C) The levels of spliced and unspliced BIC transcripts in nuclear and cytoplasmic fractions of Ramos EV and Ramos full-length spliced BIC (fl-exBIC). For both Ramos EV and fl-exBIC, unspliced BIC showed significantly higher (p<0.001) levels in the nucleus than in the cytoplasm. Spliced BIC was not detectable (ND) in Ramos EV. Upon overexpression of the fl-exBIC construct, spliced BIC transcripts showed similar levels in the cytoplasm and in the nucleus. Transcript levels in the cytoplasm and the nucleus were calculated relative to the total fraction and corrected for the amount of RNA per cell. Average of three independent experiments was presented. P values were determined by 2-way ANOVA and Bonferroni posttest (** p<0.01, ns - not significant). Relative amounts of unspliced (D) and spliced (E) BIC transcripts in DGCR8-IP compared to IgG-IP in Jiyoye, Ramos fl-exBIC and U-HO1 fl-exBIC cells. Endogenous unspliced, endogenous spliced and exogenous spliced BIC transcripts were significantly enriched in DGCR8-IP compared to IgG-IP. (F) No enrichment of HPRT transcript was observed in DGCR8-IP. Amounts were calculated relative to tRNA-Lys levels. P values were determined by Student’s t-test (** p<0.01, *** p<0.001, **** p<0.0001, ns - not significant).

https://doi.org/10.1371/journal.pone.0076647.g003

Next, we determined the subcellular localization of BIC transcripts in Ramos cells transduced with fl-exBIC (Figure 3C). Purity of cytoplasmic and nuclear fractions was validated with tRNA-Lys and U3, respectively (Fig. S2DE). In empty vector control cells, and in cells with overexpressed fl-exBIC, localization of endogenous unspliced BIC transcripts was predominantly nuclear. The endogenous spliced BIC transcripts were not detectable in empty vector control cells. The amount of the overexpressed fl-exBIC transcripts was similar in the cytoplasmic and nuclear fractions. Overexpression of fl-exBIC was followed by a strong induction of miR-155 (Figure 3B), indicating that upon overexpression nuclear spliced BIC transcripts can also serve as the primary miR-155 transcript.

To show that exogenous spliced BIC is processed to miR-155, we determined whether it is a direct target of the Microprocessor complex. We performed immunoprecipitation of DGCR8 [27] in wild-type Jiyoye, Ramos fl-exBIC and U-HO1 fl-exBIC cells (Figure 3D-F and Figure S3A-C). We assessed the relative amounts of unspliced and spliced BIC transcripts in the DGCR8 immunoprecipitation fraction (DGCR8-IP) compared to the control IgG-IP. Unspliced BIC transcript was significantly more enriched in DGCR8-IP fraction of all three cell lines, albeit at variable levels (Figure 3D). Similarly, the endogenous and exogenous spliced BIC transcripts were significantly enriched in DGCR8-IP fraction of Jiyoye, U-HO1 fl-exBIC and Ramos fl-exBIC cells (Figure 3E). The HPRT transcript used as negative control was not enriched in the DGCR8-IP fractions (Figure 3F). Enrichment of endogenous and exogenous spliced BIC in the DGCR8-IP fractions indicate that the spliced primary transcript can be used for miR-155 processing.

The unspliced/spliced ratio of BIC transcripts changes upon cellular activation

To investigate whether the ratio between unspliced and spliced BIC transcripts is altered upon induction of BIC, we activated three B-cell lymphoma cell lines using PMA/Ionomycin. Activation of DG-75, L428 and KM-H2 cells resulted in a 3- to 13-fold increase in miR-155 levels (Figure 4A). Induction of unspliced BIC transcript levels showed a 1.6 to 5.5 fold increase, whereas spliced BIC transcript levels showed a 5.4 to 31 fold increase compared to untreated cells (Figure 4B). Although the unspliced BIC transcript remained the predominant transcript, the unspliced/spliced BIC transcript ratio significantly changed in favour of the spliced BIC transcript (Figure 4C). Thus, both processing of unspliced BIC transcript to miR-155 and to spliced BIC transcripts are enhanced upon activation-induced expression of BIC. In addition, we also assessed induction of mature miR-146a and miR-22 as well as two intronic miRNAs (miR-191, miR-16) and one intergenic miRNA (let-7a). Induction of mature miRNA levels upon PMA/Ionomycin stimulation was observed only for the three exonic miRNAs (Figure S4). Both unspliced and spliced pri-miR-146a and pri-miR-22 transcript levels were induced upon PMA/Ionomycin treatment (Figure S4), however, no difference was observed for the unspliced/spliced transcripts ratios (Figure S4). Thus, the change in unspliced/spliced transcript ratio upon stimulation is specific for the BIC transcript.

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Figure 4. Induction of unspliced and spliced BIC transcripts upon cellular activation.

Fold induction in miR-155 (A), unspliced BIC and spliced BIC transcript (B) levels upon PMA/Ionomycin (P/I) treatment. Levels of miR-155 and BIC transcripts in untreated cells were averaged and fold induction upon P/I treatment was calculated. In all three cell lines, induction of spliced BIC was higher than of unspliced BIC transcripts. (C) Unspliced BIC transcripts were predominant in both untreated and P/I-treated cells, however unspliced/spliced BIC ratio significantly decreased upon P/I treatment. Sum of spliced and unspliced BIC transcript levels was set as 100%. Average of three (DG-75 and L428) or four (KM-H2) experiments was presented. P values were determined by a Student’s t-test (* p<0.05, ** p<0.01, ns - not significant).

https://doi.org/10.1371/journal.pone.0076647.g004

Cell lines and treatment

Burkitt lymphoma cell lines (Ramos, DG-75, ST486, NAMALWA, Raji, Jiyoye) were purchased from ATCC (ST486) and DSMZ (other cell lines). Diffuse large B-cell lymphoma cell lines (OCI-Ly3, SU-DHL-4, SU-DHL-6, VER) were a kind gift of A. Rosenwald (University of Würzburg, Germany) (OCI-Ly3) [46,47], A. Epstein (UCLA, CA) (SU-DHL-4 and SU-DHL-6) [46,47] or were established in our laboratory (VER) [48]. Hodgkin lymphoma cell lines (L540, L591, L1236, DEV, KM-H2, HDLM-2, L428, U-HO1) were purchased from DSMZ (L540, KM-H2), were a kind gift of V. Diehl (University of Cologne, Germany) (L591 [49], L1236 [50], HDLM-2 [49], L428 [51,52]) and P. Möller (University of Ulm, Germany) (U-HO1) [53] or were established in our laboratory (DEV) [54]. Primary mediastinal B-cell lymphoma cell lines (KARPAS-1106P, MEDB-1) [55] were a kind gift of M. Dyer (University of Leicester, UK). Cell lines were cultured at 37°C under an atmosphere containing 5% CO₂ in Iscove's Modified Dulbecco's Medium (OCI-Ly3) or RPMI-1640 (other cell lines) medium (Cambrex Biosciences, Walkersville, USA) supplemented with ultraglutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml; Cambrex Biosciences), and 5% (L428), 20% (DEV, ST486, OCI-Ly3) or 10% (other cell lines) fetal calf serum (Cambrex Biosciences). DG-75, L428 and KM-H2 cells were treated for 24h with Phorbol 12-myristate 13-acetate (PMA)/Ionomycin (both Sigma-Aldrich, Saint Louis, MO) as previously described [25]. The PMA/Ionomycin treatment was performed in triplicate (DG-75 and L428) or quadruplicate (KM-H2).

BIC/miR-155 constructs

The pcDNA3.1(+) plasmid containing full-length spliced BIC (fl-exBIC) was described previously [25]. The MXW-PGK-IRES-GFP vector was a kind gift from C-Z. Chen (Stanford University, CA). The full-length spliced fl-exBIC insert was subcloned from the pcDNA3.1(+) vector to the MXW-PGK-IRES-GFP vector using PmeI (pcDNA3.1(+) vector) and HpaI (MXW-PGK-IRES-GFP vector) restriction enzymes. The miR-155 stem-loop and ~150nt flanking sequences were amplified from genomic DNA (s-intBIC) or cDNA (s-exBIC) using Taq polymerase. Primer sequences used for PCR were as follows, 5’-TGTCACCTCCAGCTTTATAACC-3’ (forward, s-intBIC), 5’-AACCTACCAGAGACCTTACC-3’ (forward, s-exBIC), 5'-GGCTTTATCATTTTTCAATCT-3 (reverse, s-intBIC and s-exBIC). An XhoI restriction site was added to the forward and an EcoRI site to the reverse primer to allow efficient cloning. PCR products were cloned to the retroviral MXW-PGK-IRES-GFP vector using standard laboratory procedures. The inserts were sequenced to confirm the correct sequences.

Retroviral transduction

To generate retroviral particles, Phoenix-Ampho packaging cells [56] were CaPO₄ transfected with 37,5μg of MXW-PGK-IRES-GFP constructs (empty vector or vector containing one of the BIC constructs) in T75 flasks. Viral particles were harvested after two days and concentrated with Retro-X concentrator (Clontech, Saint-Germain-en-Laye, France) according to the manufacturer’s protocol. Target cells were transduced with the virus by spinning at 2,000rpm for 2hrs. Cells transduced with retroviral vectors were sorted for GFP using MoFlo sorter (Dako cytomation).

RNA isolation from total, nuclear and cytoplasmic fractions

Nuclear and cytoplasmic fractions were separated by adding 200µl of lysis buffer (140mM NaCl, 1.5mM MgCl₂, 10mM Tris-HCl pH8.0, 1mM DTT, 0.5% Nonidet P-40) to pellets of ~4 million cells, followed by 5min incubation on ice and centrifugation for 3min at 4°C and 100xg. The supernatant was harvested as the cytoplasmic fraction. The pellet containing the nuclei was washed twice with lysis buffer. 1ml of Qiazol (Qiagen, Carlsbad, USA) was added to the ~200µl of cytoplasmic fraction, to the nuclear pellet and to the total cell pellet.

Quantitative RT-PCR

Total RNA was isolated using Trizol (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol for the cell lines. RNA samples were treated with DNase (Ambion, Foster City, CA). The RNA concentration was measured with a NanoDrop^TM 1000 Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA) and RNA integrity was evaluated by 1% agarose electrophoresis. cDNA was synthesized using 500ng input RNA, SuperScript II and random primers according to the manufacturers protocol (Invitrogen). The qPCR reaction contained SYBRgreen mix (Applied Biosystems, Foster City, USA), 300nM primers, and 1ng of cDNA in a total volume of 10µl. Levels of spliced, unspliced and total BIC as well as spliced and unspliced pri-miR-22, pri-miR-146a were normalized to HPRT. For cytoplasmic, nuclear and total fractions, qRT-PCR was performed as described above with slight modifications. We used the miRNeasy kit (Qiagen) including a DNase-treatment (Qiagen) for RNA isolation. RNA was diluted in the same way for total and cytoplasmic fractions and 5 times less for nuclear fraction to use similar amounts (~500ng) for cDNA synthesis. 1/500 of the cDNA was used for qRT-PCR. C_t values of nuclear fractions were corrected for different RNA dilutions. Transcript levels in nuclear and cytoplasmic fraction were calculated relative to total fraction. Purity of nuclear fractions was confirmed with U3 and Xist and of the cytoplasmic fraction with tRNA-Lys. For qRT-PCR the following primer sequences used were: for unspliced BIC, 5’- AGCTTTATAACCGCATGTGCATAC-3’ (forward) and 5’- CAGATTTCCCCTTCCTGGTTT-3’ (reverse); for total BIC, 5’- AAATTCTTTATGCCTCATCCTCTGA-3’ (forward) and 5’-AGGCAAAAACCCCTATCACGAT-3’ (reverse); for unspliced pri-miR-22, 5’-CTGCTCAGATCTTTCCCATTTTC-3’ (forward) and 5’-CCAGGTGAGGGCGTGAGA-3’ (reversed); for spliced pri-miR-22, 5’-GGGCTGATCACTGAACTCACATT-3’ (forward) and 5’-TGAGGGCGTGAGAGGAACA-3’ (reversed), for unspliced pri-miR-146a, 5’-ATTTACCAGGCTTTTCACTCTTGTATT-3’ (forward) and 5’-GGCTTTTCAGAGATGGTGCAA-3’ (reverse); for spliced pri-miR-146a, 5’-GACAGGAGACAGTAGCACAACGA-3’ (forward) and 5’-CAGCCAGCGAGCTCCTAAAA-3’ (reverse); for Xist, 5’-GTCCTTTCTTTTGACCCCAGAA-3’ (forward) and 5’-GAGCCTGGCACTTTTTTTTCC-3’ (reverse); primers for spliced BIC, HPRT, tRNA-Lys and U3 were described previously [21,57,58]. Localization of unspliced or spliced transcript-specific qRT-PCR products are indicated in Figure 2A. qRT-PCR for miR-155, miR-22, miR-146a, miR-16, miR-191, let-7a and RNU48 was performed using miRNA qRT-PCR assays (Applied Biosystems, Foster City, USA) as described previously [59]. Reverse transcription (RT) primers specific for a miRNA and RNU48 (control) were multiplexed in 15µl RT reactions containing 1µl of each RT primer. The miRNA levels were normalized to the RNU48 levels. Mean cycle threshold (C_t) values for all genes were quantified with the SDS software (version 2.1). Relative expression levels were calculated as 2^-∆Ct.

DGCR8 immunoprecipitation

Immunoprecipitation of DGCR8-containing Microprocessor complexes was performed as described previously for Ago2-IP with some slight modifications [60]. Briefly, ~30 million cells were lysed with 100 µl of the same lysis buffer as used for the cellular fractionation and sonicated for 5 seconds. Lysate was incubated with protein A Sepharose beads (GE Healthcare) coated with anti-DGCR8 antibody (ab90579, Abcam, Cambridge, UK) at 4°C overnight. Mouse IgG antibody was used as a negative control (Millipore BV, Amsterdam, The Netherlands). After washing the beads, RNA was harvested for qRT-PCR analysis and protein lysates were prepared for Western blot. Western blot for DGCR8 was performed as described previously [60], using a 1:2000 dilution of the DGCR8 antibody at 4°C overnight. Total RNA was isolated with miRNeasy Kit (Qiagen) according to manufacturer’s protocol. RNA from DGCR8-IP and IgG-IP fractions of Jiyoye, U-HO1 fl-exBIC and Ramos fl-exBIC cells was used for qRT-PCR analysis. Levels of unspliced and spliced BIC transcripts in DGCR8-IP and IgG-IP fraction were calculated relative to tRNA-Lys and fold increase in the DGCR8-IP fraction was calculated for each sample.