Limits...
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.

Show MeSH

Related in: MedlinePlus

The snap-velcro model and supporting biochemical and genomic evidence.(A) A snap-velcro model for priming of L1 reverse transcription. The snap represents the 4 last nucleotides of the primer. It is considered as closed if it ends with 4 Ts (perfect terminal match) and as open if it contains a mismatch in the last 4 Ts. The velcro represents the 6 upstream bases. It is considered as tightly fastened only if the position-weighted T-score of this region is at least 50% of the maximum score. Otherwise, it is considered as loosely or not fastened. When the snap is closed and the velcro is tightly fastened, reverse transcription is high (bottom). If the snap is open or if the velcro is loosely fastened, reverse transcription priming is reduced (middle). Finally, if the snap is open and the velcro loosely fastened, reverse transcription priming is low or  (top). (B) In vitro efficiency of reverse transcription priming by the hL1 RNP depending of the snap and velcro status. Bars indicate the mean and error bars the S.E.M. Data are from Figure 4A, white bars excluded (see legend Figure 4). Both snap and velcro contribute extremely significantly to the differences of extension between primers (p<0.0001, two-way ANOVA). (C) Proportion of sites in the snap and velcro categories for the human genome (hg19), the repeat-masked human genome (hg19 RM) and in polymorphic L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012). Note that the proportion of sites falling in each of the snap-velcro category is significantly different in the L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012) as compared to the proportions found in hg19 or repeatmasked hg19 (Chi-square test, two-tailed P<0.0001). (D) Human L1s preferentially insert into target sites with snap closed and velcro fastened. Potential (hg19 or hg19 RM) or real (dbRIP or Lee 2012) target sites with a recognizable EN target sequence were categorized based on their snap and velcro states. The frequency of each category for each dataset was calculated and divided by the frequency of the corresponding category in the reference genome hg19 (enrichment). For each dataset, enrichment was further normalized to the enrichment of the “open snap/loose velcro” category to evaluate the respective effect of the snap and/or velcro on L1 insertion site frequencies (normalized frequency). The raw data for panels C and D are compiled in Table S2.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3649969&req=5

pgen-1003499-g005: The snap-velcro model and supporting biochemical and genomic evidence.(A) A snap-velcro model for priming of L1 reverse transcription. The snap represents the 4 last nucleotides of the primer. It is considered as closed if it ends with 4 Ts (perfect terminal match) and as open if it contains a mismatch in the last 4 Ts. The velcro represents the 6 upstream bases. It is considered as tightly fastened only if the position-weighted T-score of this region is at least 50% of the maximum score. Otherwise, it is considered as loosely or not fastened. When the snap is closed and the velcro is tightly fastened, reverse transcription is high (bottom). If the snap is open or if the velcro is loosely fastened, reverse transcription priming is reduced (middle). Finally, if the snap is open and the velcro loosely fastened, reverse transcription priming is low or (top). (B) In vitro efficiency of reverse transcription priming by the hL1 RNP depending of the snap and velcro status. Bars indicate the mean and error bars the S.E.M. Data are from Figure 4A, white bars excluded (see legend Figure 4). Both snap and velcro contribute extremely significantly to the differences of extension between primers (p<0.0001, two-way ANOVA). (C) Proportion of sites in the snap and velcro categories for the human genome (hg19), the repeat-masked human genome (hg19 RM) and in polymorphic L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012). Note that the proportion of sites falling in each of the snap-velcro category is significantly different in the L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012) as compared to the proportions found in hg19 or repeatmasked hg19 (Chi-square test, two-tailed P<0.0001). (D) Human L1s preferentially insert into target sites with snap closed and velcro fastened. Potential (hg19 or hg19 RM) or real (dbRIP or Lee 2012) target sites with a recognizable EN target sequence were categorized based on their snap and velcro states. The frequency of each category for each dataset was calculated and divided by the frequency of the corresponding category in the reference genome hg19 (enrichment). For each dataset, enrichment was further normalized to the enrichment of the “open snap/loose velcro” category to evaluate the respective effect of the snap and/or velcro on L1 insertion site frequencies (normalized frequency). The raw data for panels C and D are compiled in Table S2.

Mentions: To illustrate these findings, we propose that the four terminal bases of the primer, which overlap with the EN nuclease recognition sequence, act as a specific snap and the upstream six bases act as a weaker velcro strap (Figure 5A). When the snap is closed (perfect terminal matches, EN consensus sequence), initiation is efficient, but is enhanced if the velcro strap (upstream bases) is also tightly fastened. Inversely, if the snap is open (terminal mismatches), extension occurs preferentially if this is compensated by a tightly fastened velcro strap. The rational to distinguish snap and velcro regions is to highlight the preponderant role of the terminal nucleotides, which is also reflected in the position-weighted T-density mode of calculation.


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)

The snap-velcro model and supporting biochemical and genomic evidence.(A) A snap-velcro model for priming of L1 reverse transcription. The snap represents the 4 last nucleotides of the primer. It is considered as closed if it ends with 4 Ts (perfect terminal match) and as open if it contains a mismatch in the last 4 Ts. The velcro represents the 6 upstream bases. It is considered as tightly fastened only if the position-weighted T-score of this region is at least 50% of the maximum score. Otherwise, it is considered as loosely or not fastened. When the snap is closed and the velcro is tightly fastened, reverse transcription is high (bottom). If the snap is open or if the velcro is loosely fastened, reverse transcription priming is reduced (middle). Finally, if the snap is open and the velcro loosely fastened, reverse transcription priming is low or  (top). (B) In vitro efficiency of reverse transcription priming by the hL1 RNP depending of the snap and velcro status. Bars indicate the mean and error bars the S.E.M. Data are from Figure 4A, white bars excluded (see legend Figure 4). Both snap and velcro contribute extremely significantly to the differences of extension between primers (p<0.0001, two-way ANOVA). (C) Proportion of sites in the snap and velcro categories for the human genome (hg19), the repeat-masked human genome (hg19 RM) and in polymorphic L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012). Note that the proportion of sites falling in each of the snap-velcro category is significantly different in the L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012) as compared to the proportions found in hg19 or repeatmasked hg19 (Chi-square test, two-tailed P<0.0001). (D) Human L1s preferentially insert into target sites with snap closed and velcro fastened. Potential (hg19 or hg19 RM) or real (dbRIP or Lee 2012) target sites with a recognizable EN target sequence were categorized based on their snap and velcro states. The frequency of each category for each dataset was calculated and divided by the frequency of the corresponding category in the reference genome hg19 (enrichment). For each dataset, enrichment was further normalized to the enrichment of the “open snap/loose velcro” category to evaluate the respective effect of the snap and/or velcro on L1 insertion site frequencies (normalized frequency). The raw data for panels C and D are compiled in Table S2.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003499-g005: The snap-velcro model and supporting biochemical and genomic evidence.(A) A snap-velcro model for priming of L1 reverse transcription. The snap represents the 4 last nucleotides of the primer. It is considered as closed if it ends with 4 Ts (perfect terminal match) and as open if it contains a mismatch in the last 4 Ts. The velcro represents the 6 upstream bases. It is considered as tightly fastened only if the position-weighted T-score of this region is at least 50% of the maximum score. Otherwise, it is considered as loosely or not fastened. When the snap is closed and the velcro is tightly fastened, reverse transcription is high (bottom). If the snap is open or if the velcro is loosely fastened, reverse transcription priming is reduced (middle). Finally, if the snap is open and the velcro loosely fastened, reverse transcription priming is low or (top). (B) In vitro efficiency of reverse transcription priming by the hL1 RNP depending of the snap and velcro status. Bars indicate the mean and error bars the S.E.M. Data are from Figure 4A, white bars excluded (see legend Figure 4). Both snap and velcro contribute extremely significantly to the differences of extension between primers (p<0.0001, two-way ANOVA). (C) Proportion of sites in the snap and velcro categories for the human genome (hg19), the repeat-masked human genome (hg19 RM) and in polymorphic L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012). Note that the proportion of sites falling in each of the snap-velcro category is significantly different in the L1 insertion datasets (dbRIP, Solyom 2012 and Lee 2012) as compared to the proportions found in hg19 or repeatmasked hg19 (Chi-square test, two-tailed P<0.0001). (D) Human L1s preferentially insert into target sites with snap closed and velcro fastened. Potential (hg19 or hg19 RM) or real (dbRIP or Lee 2012) target sites with a recognizable EN target sequence were categorized based on their snap and velcro states. The frequency of each category for each dataset was calculated and divided by the frequency of the corresponding category in the reference genome hg19 (enrichment). For each dataset, enrichment was further normalized to the enrichment of the “open snap/loose velcro” category to evaluate the respective effect of the snap and/or velcro on L1 insertion site frequencies (normalized frequency). The raw data for panels C and D are compiled in Table S2.
Mentions: To illustrate these findings, we propose that the four terminal bases of the primer, which overlap with the EN nuclease recognition sequence, act as a specific snap and the upstream six bases act as a weaker velcro strap (Figure 5A). When the snap is closed (perfect terminal matches, EN consensus sequence), initiation is efficient, but is enhanced if the velcro strap (upstream bases) is also tightly fastened. Inversely, if the snap is open (terminal mismatches), extension occurs preferentially if this is compensated by a tightly fastened velcro strap. The rational to distinguish snap and velcro regions is to highlight the preponderant role of the terminal nucleotides, which is also reflected in the position-weighted T-density mode of calculation.

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