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. 2013 Oct;20(10):1173-81.
doi: 10.1038/nsmb.2658. Epub 2013 Sep 1.

The Microprocessor controls the activity of mammalian retrotransposons

Sara R Heras  1 , Sara Macias, Mireya Plass, Noemí Fernandez, David Cano, Eduardo Eyras, José L Garcia-Perez, Javier F Cáceres
Affiliations

The Microprocessor controls the activity of mammalian retrotransposons

Sara R Heras et al. Nat Struct Mol Biol. 2013 Oct.

Abstract

More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.

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Figures

Figure 1
Figure 1
DGCR8 binds a constellation of transcripts from repetitive elements. (a) Pie chart showing distribution of reads mapping to repetitive elements in sense and antisense orientation. 73% of the significant clusters map to transposable elements including DNA-Transposons, LINE-1s, LTR-containing retrotransposons and SINEs. (b) Distribution of DGCR8 binding sites in a human RC-L1Hs consensus sequence (FDR <0.01). Only sense peaks with a minimum of 29 reads are represented. Schematic representation of the RC-L1 element (bottom). UTR, untranslated region; ORF, open reading frame. (c) Distribution of significant DGCR8 binding sites in a human Alu Y consensus sequence in sense orientation (FDR <0.01). (d) Amplification of Alu Y, LINE-1 and 7SK mRNAs by RT-PCR upon immunoprecipitation of endogenous DGCR8 protein. (e) Real-time Reverse Transcriptase (RT)-PCR analysis of several transposable elements (LINE-1, Alu Y, Alu S Alu J and SVA) upon immunoprecipitation of endogenous DGCR8 of one representative experiment. Grey bars represent the relative enrichment over control IgG immunoprecipitation (black bars) and data is expressed as a percentage of input for normalization purposes. In this assay, a well-known target of the Microprocessor (pri-miR-24-1) was used as an internal positive control, whereas 7SK and ACTB were used as negative controls. IP,immunoprecipitation.
Figure 2
Figure 2
The Microprocessor regulates the abundance of L1 mRNA and L1-encoded ORF1p protein. (a) Real time RT-PCR analysis of endogenous L1 mRNA in PA-1 cells upon depletion of Drosha. Western blot analysis confirms Drosha depletion (bottom panel). (b) Northern blot analysis of endogenous polyadenylated L1 mRNA in PA-1 cells upon overexpression of Dominant negative (DN) forms of both DGCR8 (lane 2) and Drosha (lane 3 (asterisks depict smaller fragments arising from cryptic splice sites or truncated versions of L1 mRNAs). Gapdh hybridization was used as a loading control. Western blot analysis shows the level of expression of the DN forms (bottom panel). (c) Real time RT-PCR analysis of mouse TF L1 mRNA in Dgcr8−/− mouse ES cells and parental ES cells (WT). (d) Western blot analysis of L1-ORF1p in Dgcr8−/− mouse ES cells and parental ES cells (WT). Western-blot was quantified using ImageQuant TL software (GE) plotted data are the average of 3 technical replicates. (a) and (c) values are averages of three independent biological replicates; *P< 0.05 (t test). Error bars, s.d. Uncropped versions of the blots are shown in Supplementary Fig. 8.
Figure 3
Figure 3
The 5′UTR of L1 mRNA is cleaved by immunopurified Drosha in vitro. (a) Schematic representation of four 300-nucleotide in vitro transcribed fragments spanning the sense L1 5′UTR region used in (b) for in vitro processing. Transcripts were incubated (+) or not (−) with immunopurified Microprocessor (using FLAG-Drosha). Cleavage products are indicated with asterisks (lanes 2, 4, 6). An RNA ladder marker indicates sizes in base-pairs on the left. (c) In vitro processing of 285-500 L1 5′UTR region (lane 2). Top panel shows a predicted pri-miRNA-like structure of this region. Cleavage is abolished upon introducing mutations that disturb this structure (lane 4 and top panel).
Figure 4
Figure 4
The Microprocessor negatively regulates L1 retrotranspositionin vivo. (a) Cartoon depicting the LINE-1 based retrotransposition assay in cultured cells. The transcription start site at the L1 5′ UTR (black arrow), the L1 open reading frames (ORF1 and ORF2; gray rectangles), and the L1 poly (A) site (grey lollipop) are indicated. The relative locations of the endonuclease (EN), reverse transcriptase (RT), and cysteine-rich (C) domains of ORF2 are also indicated. (b) Schematic representation of the JJ101(L1.3) vector containing a full-length human RC-L1 sequence tagged with a mblastI cassette (Blasticidin-resistance gene). The 5′UTR of L1.3 was removed to generate construct TAM102(L1.3). Alleles containing a missense mutation in the Reverse Transcriptase (RT) domain of L1-ORF2 (red asterisk, JJ101 (L1.3) D702A and TAM102 (L1.3) D702A) were used as an internal control. CMV denotes the presence of a Cytomegalovirus promoter. Black and grey lollipops represent polyadenylation signals. (c,e) Cell culture-based LINE-1 retrotranposition assay. (c) HeLa cells were co-transfected with JJ101(L1.3), TAM102(L1.3) and corresponding control vectors or the indicated engineered LINE-1 construct containing an individual fragment derived from the 5′UTR region plus the indicated expression plasmids (β-arrestin (β-arr), Apobec3A, or DN Drosha), as indicated on the left. Each image shows representative data from L1 retrotransposition assays conducted in triplicate. (d,f) Quantification of LINE-1 retrotranposition (from panel c and e respectively). Blasticidin-resistant foci were manually counted and quantified. Data is presented as the proportion of the activity seen in cultures co-transfected with the plasmid expressing the negative control (β-arrestin) and normalized using transfection efficiency and toxicity. Shown are averages of 3 independent biological replicates ± s.d.
Figure 5
Figure 5
Alu is in vitro processed and its retrotransposition is regulated by the Microprocessor. (a) In vitro processing of Alu Ya5. An Alu Ya5 core sequence was in vitro transcribed and incubated with Flag-Drosha immunoprecipitates. Upon addition of Drosha (+) Alu Ya5 transcripts were cleaved (lane 4), processing of pri-miR-30c-1 is shown as a positive control (lane 2). (b) Rationale of the Alu trans-mobilization assay. In the scheme, a cartoon depicts the ‘ORF2 driver’ containing an exogenous promoter (white arrow, CMV) and the coding sequence for LINE-1 ORF2 (grey rectangle). In addition, a tagged Alu (grey arrow) with a neoTet retrotransposition indicator cassette (black boxed backwards Neo) containing an encoded poly (A) tail (A33) is depicted. Note that the neoTet cassette contains a self-spliceable group I intron (black curvy lane). Lollipops represent polyadenylation signals. With this configuration, expression of a Neomycin resistant gene can only occur upon a round of trans-retrotransposition (bottom part of the panel). (c) Trans-retrotransposition experiments using 5′UTR-ORF2-NN and (d) ORF2co-NN as a driver (scheme shown). Each image shows representative data from Alutrans-retrotransposition assays conducted in duplicate. β-arrestin is used as a control as it does not affect Alu retrotransposition. The relative retrotransposition activity is quantitated on the right. Data was normalized using transfection efficiency and toxicity. Shown is average of three independent biological replicates ± s.d.
Figure 6
Figure 6
LINE-1 Regulation by the Microprocessor is Dicer and miRNA-independent. (a) Schematic representation of firefly luciferase reporters containing the 5′UTR from a mouse RC-L1-L1spa- (mL1spa) and controls, simian virus 40 promoter (SV40) and SV40-miR18a, containing a target site for miR-18a in the 3′UTR. LCS; firefly luciferase open reading frame. (b) Dgcr8−/−, Dicer−/− ES cells, as well as parental wild-type cells (v6.5 and f/f Dicer, respectively) were transfected with vectors described in (a). Error bars indicate standard deviation (n=3 biological replicas) and *P< 0.05 (t test). (c) Cartoon depicting the LINE-1 engineered constructs assayed in cultured cells (following nomenclature used in Fig. 4). Construct JJ101(L1.3)Δ3′UTR is a derivative of plasmid JJ101(L1.3) that lacks LINE-1 3′UTR. (d) HeLa cells were co-transfected with JJ101(L1.3) or JJ101(L1.3)Δ3′UTR vectors plus the indicated expression plasmids (β-arrestin (β-arr) or DN Drosha), as indicated on the left side. Each image shows representative data from L1 retrotransposition assays conducted in triplicate. The graph shows a quantification of the assay, and data is presented as the proportion of the activity seen in cultures co-transfected with the plasmid expressing the negative control (β-arrestin) and normalized using transfection efficiency and toxicity. Shown are averages of 3 independent biological replicates ± s.d.
Figure 7
Figure 7
A model for the control of LINE-1 and Alu retrotransposition by the Microprocessor. Details are provided within the text.
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References

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