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Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.

ABSTRACT: Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.

SUBMITTER: Guo LT

PROVIDER: S-EPMC9262592 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules

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Project description:The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5' end of one template to the 3' end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI-IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3' end of the acceptor template. The reliance on only a single bp with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement-encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.

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| S-EPMC2570493 | biostudies-literature

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