miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPA1 mRNA

Abstract

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1's ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability.

Keywords: LINE-1; hnRNPA1; miR-128; miRs; restriction factor; retrotransposition.

Figure 1

Identification of hnRNPA1 as a cellular target of miR-128. (A) Table showing the results of an overlay analysis of the genes identified in our mouse embryonic stem cell (mESC) DGCR8^−/− screen of putative miR-128 targets by differential gene expression and protein targets previously reported as L1 ORF1p interactome [5]. Members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) family of RNA-binding proteins are highlighted in grey, see Supplementary Table S1 for additional information. (B) Relative amount of hnRNPA1, hnRNPA2B1, hnRNPK, hnRNPL and hnRNPU and TNPO1 (positive control) mRNA normalized to B2M determined in HeLa cells stably transduced with control miR, anti-miR-128 or miR-128 are shown as mean ± SD (n = 3 technical replicates, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

miR-128 reduces hnRNPA1 mRNA and protein amounts whereas miR-128 neutralization enhances hnRNPA1 expression levels in multiple cell types. (A) Relative amount of hnRNPA1 mRNA normalized to B2M in HeLa cells stably transduced with miR-128, anti-miR-128 or control constructs (left panel, n = 2 independent biological replicates, p = ns); or transiently transfected with miR-128, anti-miR-128 or control mimics (right panel, mean ± SEM, n = 3 independent biological replicates, * p < 0.05) (B) Immunoblot analysis of hnRNPA1 and α-tubulin protein levels in lysates from HeLa cells transduced with miR-128, anti-miR-128 or miR control constructs (left panel). Quantification of blots is shown (right panel). (C) Stable miR-128, anti-miR-128 and control miR HeLa cell lines were analyzed by immunofluorescence for hnRNPA1 expression and co-localization with DAPI. (D) Relative amounts of hnRNPA1 mRNA normalized to B2M in induced pluripotent stem cells, colorectal cancer initiating cells (CCIC), breast cancer cells (MDA-MB-231), non-small cell lung cancer (A549) cells and teratoma (Tera-1) cells. (E) Immunoblot analysis of hnRNPA1 and α-tubulin protein levels in protein-containing lysates isolated from non-small cell lung cancer (A549), colon cancer (SW620) (PANC1) cells transduced with miR-128, anti-miR-128 or miR control constructs. Quantification of blots are shown (bottom panels). Throughout the figure if not otherwise noted, n = 3 independent biological replicates, mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, calculated by students t-test.

Figure 3

miR-128 represses hnRNPA1 expression by binding directly to coding sequence (CDS) RNA. (A) Schematic of the predicted miR-128 7-mer binding site in the coding region (CDS) of hnRNPA1 mRNA (top panel). (B) Cartoon showing the predicted base pairing of miR-128 to the seed sequence of wild-type (WT) hnRNPA1 as well as a representation of mutations in the seed sequence (mutant) used for luciferase binding assays (top panel). Relative luciferase activity in HeLa cells transfected with plasmids expressing a Gaussia luciferase gene fused to the wild-type (WT) binding site or positive control sequence corresponding to the 22 nucleotide perfect match of miR-128 and co-transfected with control or mature miR-128 mimics were determined 48 h post-transfection (bottom left panel, n = 3 independent biological replicates, mean ± SEM, ** p < 0.01, **** p < 0.0001). Relative luciferase activity in HeLa cells transfected with plasmids expressing the luciferase gene fused to the WT or the mutated binding site (mutant) and co-transfected with control or mature miR-128 mimics were determined 48 h post-transfection (bottom right panel, n = 3 independent biological replicates, mean ± SEM, * p < 0.05). (C) Cartoon of Argonaute-RNA immuno-purification (miRISC-IP). (D) miRISC IP of HeLa cell lines stably transduced with miR-128 overexpression or miR-128 neutralization (anti-miR-128) were performed. Relative amounts of hnRNPA1 RNA normalized to B2M were determined for input samples (top left panel “input—hnRNPA1”, n = 3 independent biological replicates, mean ± SEM, * p < 0.05). Relative fractions of hnRNPA1 transcript amounts associated with immune-purified Ago complexes are shown for immunopurified (IP) samples, hnRNPA1 fractions normalized to the amount of TNPO1 in the input are shown as “corrected” (top right panel “IP—hnRNPA1”, n = 3 independent biological replicates, mean ± SEM, * p < 0.05, ** p < 0.01). (D) Relative amount of GAPDH in the same input and IP samples were determined as a negative control (top right panel “IP—hnRNPA1”, n = 3 independent biological replicates, mean ± SEM). Throughout the figure, n = 3 independent biological replicates, mean ± SEM, * p < 0.05, ** p < 0.01, **** p < 0.0001, calculated by students t-test.

Figure 4

Overexpression of hnRNPA1 enhances de novo L1 retro-transposition and hnRNPA1 knock-down reduces L1 mobilization. (A) De novo retro-transposition was determined by a colony formation assay in HeLa cells stably transduced with plasmids encoding control plasmid (control), hnRNPA1 and transfected with L1 expression construct. Western blot analysis validating reduced levels of hnRNPA1 in over-expressing cell lines are shown. α-tubulin was used as a loading control (right panel). (n = 3 technical replicates, mean ± SD, ** p < 0.01). (B) Representative example of neomycin-resistant colony counts from a colony formation assay in HeLa cells stably transduced with plasmids encoding shRNA against GFP (Green Fluorescent Protein) (control), hnRNPA1 and transfected with WT L1 (L1) or RT deficient L1 (RT-dead L1) expression construct. (n = 3 technical replicates, mean ± SD, *** p < 0.001). Western blot analyses of hnRNPA1 and α-tubulin in knock-down HeLa cell lines are shown (right panels).

Figure 5

hnRNPA1 partly rescues miR-128-induced inhibition of de novo L1 retrotransposition. The functional importance of hnRNPA1 in miR-128-induced L1 repression was evaluated by colony formation assays using mutant L1 (miR-128 resistant) or RT deficient L1 (RT-dead L1) expression constructs in stable HeLa cell lines expressing either miR-control (miR-CTL) or miR-128 along with either a plasmid control (FL-CTL) or induced hnRNPA1 (hnRNPA1) (n = 3 technical replicates, mean ± SD, ** p < 0.01). Western blot analysis was performed for hnRNPA1 and α-tubulin to validate increased levels of hnRNPA1 in miR-128-hnRNPA1 rescue HeLa cell lines (panel).