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. 2006 Feb 6;34(3):853-64.
doi: 10.1093/nar/gkj490. Print 2006.

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition

Patrick Wai-Lun Li  1 , Jinfang Li, Stephanie L Timmerman, Les A Krushel, Sandra L Martin
Affiliations

The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition

Patrick Wai-Lun Li et al. Nucleic Acids Res. .

Abstract

Most eukaryotic mRNAs are monocistronic and translated by cap-dependent initiation. LINE-1 RNA is exceptional because it is naturally dicistronic, encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Here, we show that sequences upstream of ORF1 and ORF2 in mouse L1 function as internal ribosome entry sites (IRESes). Deletion analysis of the ORF1 IRES indicates that RNA structure is critical for its function. Conversely, the ORF2 IRES localizes to 53 nt near the 3' end of ORF1, and appears to depend upon sequence rather than structure. The 40 nt intergenic region (IGR) is not essential for ORF2 IRES function or retrotransposition. Because of strong cis-preference for both proteins during L1 retrotransposition, correct stoichiometry of the two proteins can only be achieved post-transcriptionally. Although the precise stoichiometry is unknown, the retrotransposition intermediate likely contains hundreds of ORF1ps for every ORF2p, together with one L1 RNA. IRES-mediated translation initiation is a well-established mechanism of message-specific regulation, hence, unique mechanisms for the recognition and control of these two IRESes in the L1 RNA could explain differences in translational efficiency of ORF1 and ORF2. In addition, translational regulation may provide an additional layer of control on L1 retrotransposition efficiency, thereby protecting the integrity of the genome.

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Figures

<b>Figure 1</b>
Figure 1
An IRES upstream of ORF1 and ORF2 in mouse L1. (A) Schematic of the RNA that created L1spa: tandem boxes on the left represent 7.2 copies of a 212 nt repeated motif known as the TF monomer which contains a promoter for transcription (18); these are followed by a 257 nt 5′-UTR (line); ORF1, which overlaps with ORF2 by 14 nt, although the UAA termination codon of ORF1 is separated from the first AUG in ORF2 by a 40 nt IGR; ORF2; a 3′-UTR (line) and a polyA tail (An). Note that the 5′ end of each L1 mRNA depends upon which monomer was used to initiate transcription, thus the sequences upstream of the ORF1 AUG can differ dramatically in length and base composition (monomer sequences are 65% GC, whereas the remaining 5′-UTR is 47% GC). Regions of L1spa tested for IRES activity using the dicistronic reporter gene assay are delineated. (B) Schematic of the pRF dicistronic reporter vector containing firefly (Fluc) and Renilla (Rluc) luciferase genes. The dicistronic mRNA is transcribed by an SV40 promoter/enhancer sequence in mouse cells and by a T7 promoter in vitro. Insertion of ΔEMCV upstream of the test sequence greatly reduces translation of Fluc by read-through or reinitiation from Rluc (28). (C) Relative lucificerase activity (to pRF without an insert ± SD) obtained from pRF containing 400 or 201 nt upstream of the ORF1 (400-1 UTR) or ORF2 (201-1 IGR) AUG or various sequences further 3′ in L1. These are named by their lengths and are from the 3′ end of ORF2 (202, 47, 220, 140) or the 3′-UTR (312, 191); their GC contents vary between 38 and 49%. The known Cricket Paralysis Virus intergenic IRES [CrPV (28)] was included as a positive control.
<b>Figure 2</b>
Figure 2
Northern blot analysis of Fluc RNA. Phosphorimage of northern blot of RNA recovered from DNA transfection of the dicistronic constructs containing L1 (A) or TrkB (B) sequences as indicated. Blots were hybridized to Fluc. The positions of size standards run in the adjacent lane are indicated. Full-length dicistronic transcripts are indicated by the arrows; the truncated TrkB Fluc transcript from the cryptic promoter is indicated by the open arrowhead. The coding sequence for Fluc is 1656 nt.
<b>Figure 3</b>
Figure 3
Promoterless vector assays. (A) Schematic of promoterless vector, the arrow indicates the insertion site of the test sequences. (B) Relative luciferase activities (to CrPV ± SD) of the 400 nt (400-1 UTR) or 201 nt (201-1 IGR) upstream of ORF1 and ORF2 compared with 312 (L1 3′-UTR) and mouse TrkB (exon 1 and 2) are plotted below the schematic of the promoterless vector. This region of TrkB contains a cryptic promoter (Figure 2). 312 is also devoid of activity in the dicistronic assay of Figure 1.
<b>Figure 4</b>
Figure 4
RNA transfection. Relative luciferase activity (to pRF ± SD) following transfection of RNA transcribed in vitro from pRF constructs as depicted in Figure 1.
<b>Figure 5</b>
Figure 5
Test of translational independence of the second cistron in the dicistronic reporter construct. Phosphorimages of SDS–PAGE gels containing proteins labeled with [35S]methionine during in vitro translation. Rabbit reticulocyte lysates were programmed with 20 ng/µl of dicistronic RNA transcribed in vitro by T7 polymerase as indicated. Rluc translation is controlled by either the EMCV IRES (A), or ΔEMCV (B), Fluc translation is controlled by the three indicated sequences. Results identical to these were obtained using 10 ng/µl of RNA in separate reactions.
<b>Figure 6</b>
Figure 6
Mapping the ORF1 IRES. Schematics of deletion constructs upstream of ORF1 are shown on the left. These regions of L1 were cloned into either pRF or the promoterless vector and tested for luciferase expression; relative luciferase activities are plotted after normalization to pRF (dicistrionic, middle) or to the CrPV IRES (promoterless, right). (A) 5′ truncation series, (B) 3′ truncation series, (C) 100 nt fragments and (D) 200 nt fragments as indicated.
<b>Figure 7</b>
Figure 7
Mapping the ORF2 IRES. (A) Truncations and fragments of the ORF2 IRES tested in the dicistronic reporter assay with (dicistronic, middle) and without (promoterless, right) a promoter. Relative luciferase activities are plotted as in Figure 6. (B) The indicated point mutations in the putative stem–loop tested as in (A), point mutations are underlined, the new AUG in mut 2 and the resulting in frame stop codons (UAAUAA) are boxed.
<b>Figure 8</b>
Figure 8
Rapid evolution in the IGR of L1. (A) VISTA plot () to visualize sequence similarity between mouse L1 and rat (top) or human (bottom) from the conserved region of ORF1 [amino acid 150 (55)] through the conserved endonuclease domains of ORF2 (10) including the IGR. Percent identity (between 50–100%) is plotted for 100 nt windows across the length compared, gray fills are regions with at least 60% identity for 100 nt. (B) Enlargement of the IGR of mouse L1 aligned with the homologous rat and human sequences. Sequence begins in the poorly conserved C-terminal region of ORF1 and extends through the first AUG of human ORF2. The ORF1 stop and ORF2 start codons are bold and underlined in all three sequences; the AUGs in the IGR of rat and human are bold and capitalized, as are their in frame stops which would lead to translation of 18 or 6 amino acids peptides, respectively. Both of these uORFs in the IGR require a +1 frameshift to match the frame of the ORF2 AUG.
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