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. 2010 May 1;19(9):1712-25.
doi: 10.1093/hmg/ddq048. Epub 2010 Feb 10.

Discrete subcellular partitioning of human retrotransposon RNAs despite a common mechanism of genome insertion

John L Goodier  1 , Prabhat K Mandal, Lili Zhang, Haig H Kazazian Jr
Affiliations

Discrete subcellular partitioning of human retrotransposon RNAs despite a common mechanism of genome insertion

John L Goodier et al. Hum Mol Genet. .

Abstract

Despite the immense significance retrotransposons have had for genome evolution much about their biology is unknown, including the processes of forming their ribonucleoprotein (RNP) particles and transporting them about the cell. Suppression of retrotransposon expression, together with the presence of retrotransposon sequence within numerous mRNAs, makes tracking endogenous L1 RNP particles in cells problematic. We overcame these difficulties by assaying in living and fixed cells tagged-RNPs generated from constructs expressing retrotransposition-competent L1s. In this way, we demonstrate for the first time the subcellular colocalization of L1 RNA and proteins ORF1p and ORF2p, and show their targeting together to cytoplasmic foci. Foci are often associated with markers of cytoplasmic stress granules. Furthermore, mutation analyses reveal that ORF1p can direct L1 RNP distribution within the cell. We also assayed RNA localization of the non-autonomous retrotransposons Alu and SVA. Despite a requirement for the L1 integration machinery, each manifests unique features of subcellular RNA distribution. In nuclei Alu RNA forms small round foci partially associated with marker proteins for coiled bodies, suborganelles involved in the processing of non-coding RNAs. SVA RNA patterning is distinctive, being cytoplasmic but without prominent foci and concentrated in large nuclear aggregates that often ring nucleoli. Such variability predicts significant differences in the life cycles of these elements.

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Figures

Figure 1.
Figure 1.
The structure of human retrotransposons and constructs used in this study. (A) The MS2-NLS-GFP reporter construct used for RNA-tethering. (B–F) L1 constructs. Locations of RNA FISH oligonucleotide probe sequences, α-ORF1 and α-ORF2-C epitopes, and point mutations in the two ORFs are marked. The name of the cloning vector is to the left and the exogeneous promoter is indicated when present. The cloning site for six tandem MS2-CP binding sites is marked in (A). pCEP 5′-UTR ORF2 No Neo (C) is described in ref. . p(A): poly (A) signal. (G) Alus from two subfamilies, Ya5 and AluSx, were used in the study. They differed in their terminator sequences, and were either tagged with 6X MS2-CP binding sites or left wild-type. The A- and B-box sequences of the internal pol III promoter are marked. TTTT: pol (III) terminator. (H) SVASPTA1. The vector is pcDNA6 myc/hisB (Invitrogen), containing or lacking CMV promoter. (I) Northern blot analysis showing that the expression of an MS2-CP stem loop-tagged L1 (Lane 3) does not differ from that of untagged L1 (Lane 2) in transfected HEK 293T cells. Lane 1: CEP-PUR (99-PUR with CMV promoter) vector only. A riboprobe extending from the L1 poly (A) signal to the SV40 poly (A) signal of 99-PUR L1-RP (A) was labeled with digioxygenin by T7 polymerase transcription.
Figure 2.
Figure 2.
The subcellular localization of exogenously-expressed 99-PUR L1-RP MS2 and 99-PUR L1-RP (no MS2-CP binding sites) RNA in HEK 293T cells. (A) MS2-NLS-GFP fusion protein is nuclear when expressed alone. (B) L1-MS2 RNA detected in the cytoplasm and in granules by MS2-NLS-GFP tethering. Nomarski imaging is shown and phase-dense cytoplasmic granules are marked with arrows. (C) L1-MS2 RNA detected by RNA FISH using the Cy3-MS2 oligonucleotide probe. The nuclear membrane is outlined. (D) Colocalization in the cytoplasm and granules (shown by arrows) of L1-MS2 RNA detected by MS2-NLS-GFP tethering and ORF1p detected by α-ORF1p immunofluorescence. (E) L1 RNA lacking MS2 stem loops fails to bind MS2-NLS-GFP protein in the cytoplasm. (F–H) Association of L1-MS2 RNA, detected by MS2-NLS-GFP protein binding (F) or RNA FISH using the Cy3-MS2 probe (G, H), and the stress granule marker protein TIA-1. Overlapping ORF1p and TIA-1 foci are marked by arrows. ORF1p foci not marked by TIA-1 are indicated by open triangles. L1 RNA foci do not significantly colocalize, but can be adjacent with Dcp2 (I) or GW182 (J) protein markers of processing bodies (indicated by arrows and shown in enlargements). The color scheme of all figure panels follows that of the fluorophores used for detection (green for GFP, 6-FAM and Cy2, red for Cy3, blue for Cy5).
Figure 3.
Figure 3.
ORF2 protein detected in the L1 RNP particle in HEK 293T cells. (A) ORF2p expressed alone from construct pCEP 5′UTR ORF2 No Neo and detected by α-ORF2-C. (B) Wild-type ORF2p (Lane 2) expressed with the MVA/T7RP system (19) in 143Btk- osteosarcoma cells is detected as a single prominent band of approximately 150 kDa by α-ORF2-C and western blotting. Lane 1: vector only. (C) Confocal micrograph of EGFP-tagged ORF1p, ORF2p detected by α-ORF2-C, and L1 RNA detected by the Cy5-SV40-2 FISH probe expressed from the same ORF1-EGFP L1-RP WT construct. (D) When expressed from the ORF1(1-130)-EGFP L1-RP EN RT MT construct, ORF1p deleted at the C-terminal and lacking the RNA-binding domain fails to direct ORF2p to cytoplasmic granules. (E, F) However, ORF2p and N-terminal-deleted ORF1p expressed from ORF1(131-339)-EGFP L1-RP EN RT MT exactly colocalize in the cytoplasm and nucleus. Scale bar, 10 μm.
Figure 4.
Figure 4.
The subcellular localization of exogenously-expressed Alu RNA. (A–C) MS2-tagged pBS 7SL Alu-MS2 (Ya5) Alu RNA detected by the Cy3-MS2 probe in different human cell lines. Alu nuclear foci (selectively indicated by arrows) appear phase-dense in the accompanying Nomarski images. Nuclei have been outlined in Panel B. Confocal micrographs showing that Alu RNA is sequestered in cytoplasmic granules of 293T cells (D) by coexpressed EGFP-tagged ORF1 protein, but not (E) by ORF2 protein (expressed from construct pCEP 5′UTR ORF2 No Neo). Alu RNA detected in 293T cells by MS2-NLS-GFP protein binding does not colocalize with the proteins (F) nucleophosmin marking nucleoli (a multinucleated cell is shown), (G) SC-35 marking nuclear speckles, (H) PTB marking perinucleolar compartments, or (I) Ki67. However, a subset of Alu RNA foci juxtapose with Cajal bodies (J, K) marked by SMN protein, or (L) p80-coilin. (M) Some Alu RNA foci also associate with PML bodies. Scale bar, 10 μm.
Figure 5.
Figure 5.
The subcellular localization of exogenously-expressed SVA RNA in 293T cells. (A) MS2-CP-tagged full-length pcDNA SVASPTA1 MS2 RNA detected by the Cy3-MS2 DNA probe. (B) Northern blot analysis of full-length SVASPTA1 RNA expressed (a) without, or (b) with a CMV promoter, detected by an αα-P32-labeled DNA probe spanning the MS2-CP binding repeats. (c) empty vector. SVA nuclear aggregates do not colocalize with proteins marking (C) Cajal bodies, or (D) PML bodies. (E) Differential fluorescent FISH-labeling shows Alu RNA foci (marked by arrows) to be juxtaposed but excluded from SVA nuclear aggregates.
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