Figure 1

Classical target primed reverse transcription (TPRT), twin priming, variants of twin priming and dual priming mechanisms. (a) A schematic of classical TPRT. The poly(A) tail of an L1 mRNA anneals to the target site created by L1 endonuclease. L1 reverse transcription (RT) primes at the target site and synthesizes the bottom-strand cDNA. A subsequent second-strand nick and synthesis results in an L1 insertion with a 3' poly(A) flanked by TSDs. (b) Twin priming. In this variant of TPRT, after the second-strand nick, a site internal to the mRNA anneals to the top strand overhang. A second RT molecule primes at this site, generating an inverted L1 cDNA. (c) This twin priming variant involves the disengagement of the first RT before reaching the end of the poly(A) tail, resulting in an insertion with a 5' poly(T) stretch, but lacking a 3' poly(A) tail. Like classical twin priming, this mechanism results in an inverted L1 structure. (d) A second twin priming variant creates an insertion with both a 3' poly(A) tail and a 5' poly(T) stretch. The first RT falls off before reaching the end of the poly(A) tail. (e) Dual priming. Classical TPRT involving the first mRNA begins on the first strand. After the second strand nick, a second mRNA anneals to the second strand and undergoes classical TPRT. Note that this panel is rotated 180° relative to the orientation of all other panels. This is done to show that the resulting insertion will appear the same to computational filters as the above twin priming variant.