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The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Monot C, Kuciak M, Viollet S, Mir AA, Gabus C, Darlix JL, Cristofari G - PLoS Genet. (2013)

Bottom Line: Third, efficient priming in the context of duplex DNA requires a 3' overhang.This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription.Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1081, Institute for Research on Cancer and Aging, Nice, Nice, France.

ABSTRACT
L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

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Double-stranded primers with blunt or 3′-recessed are not efficiently extended by mL1 RNPs.(A) Scheme of the primers used. (B) DLEA showing the absence of extension of double-stranded primers with blunt or 3′ recessed ends in the presence of α-32P-dTTP. Note that the only products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities (see text and Figure 8 for further characterization). RC denotes a 30 nt recovery control added after the reaction but before DNA purification. Quantification of primer extension (% Activity) was relative to levels of extension obtained with ext-(dT)18 (lane 2). The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Results obtained with hL1 RNPs were identical and are shown in Figure S4.
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pgen-1003499-g006: Double-stranded primers with blunt or 3′-recessed are not efficiently extended by mL1 RNPs.(A) Scheme of the primers used. (B) DLEA showing the absence of extension of double-stranded primers with blunt or 3′ recessed ends in the presence of α-32P-dTTP. Note that the only products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities (see text and Figure 8 for further characterization). RC denotes a 30 nt recovery control added after the reaction but before DNA purification. Quantification of primer extension (% Activity) was relative to levels of extension obtained with ext-(dT)18 (lane 2). The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Results obtained with hL1 RNPs were identical and are shown in Figure S4.

Mentions: (A) Relative extension of primers as measured by DLEA. Extension of each primer was normalized to the extension levels obtained with the (dT)20 primer (LOU541 corresponding to the 10BglIIL1.3 insertion site). This ratio, expressed as a percentage, was designated as ‘Relative activity’. Bars were color-coded and sorted according to the efficiency of priming (red, activity ≥80%; medium red, 40%≤Activity<80%; light red, activity <40%; white, primers excluded from the correlation analyses due to hairpin formation). Bars indicate the mean and error bars the S.E.M. (n = 3). The name of the insertion sites correspond to the recovered clones from cultured cells published in [46]. (B) A role for the primer terminal nucleotides in hL1 RNP reverse transcription priming. For each n between 1 and 20, the correlation between activity and position-weighted T-density of the terminal n nucleotides was calculated. The goodness-of-fit (R2) only marginally changes when n>10, indicating that the terminal 10 nucleotides are the most relevant determinants for priming efficiency. Note that the 4th bases at the 3′ terminus in all the primers of this set are coincidentally identical (T). For this reason, R2 is identical for n = 3 and n = 4. See the ‘Results’ and ‘Material and Methods’ sections for a detailed definition of the position-weighted T-density. (C) An example of correlation between the density of Ts close to the 3′ end of the primer (position-weighted T-density) and the efficiency of reverse transcription priming (for n = 10). For the graph shown in (B) and (C), primers which could fold into a structured hairpin (white bars in A) were excluded from the analysis (see Figure 6, Figure 7, Figure 8 for a detailed analysis of primer structure on reverse transcription efficiency).


The specificity and flexibility of l1 reverse transcription priming at imperfect T-tracts.

Monot C, Kuciak M, Viollet S, Mir AA, Gabus C, Darlix JL, Cristofari G - PLoS Genet. (2013)

Double-stranded primers with blunt or 3′-recessed are not efficiently extended by mL1 RNPs.(A) Scheme of the primers used. (B) DLEA showing the absence of extension of double-stranded primers with blunt or 3′ recessed ends in the presence of α-32P-dTTP. Note that the only products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities (see text and Figure 8 for further characterization). RC denotes a 30 nt recovery control added after the reaction but before DNA purification. Quantification of primer extension (% Activity) was relative to levels of extension obtained with ext-(dT)18 (lane 2). The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Results obtained with hL1 RNPs were identical and are shown in Figure S4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3649969&req=5

pgen-1003499-g006: Double-stranded primers with blunt or 3′-recessed are not efficiently extended by mL1 RNPs.(A) Scheme of the primers used. (B) DLEA showing the absence of extension of double-stranded primers with blunt or 3′ recessed ends in the presence of α-32P-dTTP. Note that the only products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities (see text and Figure 8 for further characterization). RC denotes a 30 nt recovery control added after the reaction but before DNA purification. Quantification of primer extension (% Activity) was relative to levels of extension obtained with ext-(dT)18 (lane 2). The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Results obtained with hL1 RNPs were identical and are shown in Figure S4.
Mentions: (A) Relative extension of primers as measured by DLEA. Extension of each primer was normalized to the extension levels obtained with the (dT)20 primer (LOU541 corresponding to the 10BglIIL1.3 insertion site). This ratio, expressed as a percentage, was designated as ‘Relative activity’. Bars were color-coded and sorted according to the efficiency of priming (red, activity ≥80%; medium red, 40%≤Activity<80%; light red, activity <40%; white, primers excluded from the correlation analyses due to hairpin formation). Bars indicate the mean and error bars the S.E.M. (n = 3). The name of the insertion sites correspond to the recovered clones from cultured cells published in [46]. (B) A role for the primer terminal nucleotides in hL1 RNP reverse transcription priming. For each n between 1 and 20, the correlation between activity and position-weighted T-density of the terminal n nucleotides was calculated. The goodness-of-fit (R2) only marginally changes when n>10, indicating that the terminal 10 nucleotides are the most relevant determinants for priming efficiency. Note that the 4th bases at the 3′ terminus in all the primers of this set are coincidentally identical (T). For this reason, R2 is identical for n = 3 and n = 4. See the ‘Results’ and ‘Material and Methods’ sections for a detailed definition of the position-weighted T-density. (C) An example of correlation between the density of Ts close to the 3′ end of the primer (position-weighted T-density) and the efficiency of reverse transcription priming (for n = 10). For the graph shown in (B) and (C), primers which could fold into a structured hairpin (white bars in A) were excluded from the analysis (see Figure 6, Figure 7, Figure 8 for a detailed analysis of primer structure on reverse transcription efficiency).

Bottom Line: Third, efficient priming in the context of duplex DNA requires a 3' overhang.This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription.Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1081, Institute for Research on Cancer and Aging, Nice, Nice, France.

ABSTRACT
L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.

Show MeSH
Related in: MedlinePlus