SELEX and SHAPE reveal that sequence motifs and an extended hairpin in the 5’ portion of Turnip crinkle virus satellite RNA C mediate fitness in plants

Abstract

Noncoding RNAs use their sequence and/or structure to mediate function(s). The 5' portion (166 nt) of the 356-nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) was previously modeled to contain a central region with two stem-loops (H6 and H7) and a large connecting hairpin (H2). We now report that in vivo functional selection (SELEX) experiments assessing sequence/structure requirements in H2, H6, and H7 reveal that H6 loop sequence motifs were recovered at nonrandom rates and only some residues are proposed to base-pair with accessible complementary sequences within the 5' central region. In vitro SHAPE of SELEX winners indicates that the central region is heavily base-paired, such that along with the lower stem and H2 region, one extensive hairpin exists composing the entire 5' region. As these SELEX winners are highly fit, these characteristics facilitate satRNA amplification in association with TCV in plants.

Introducing SELEX via a semester‐long course‐based undergraduate research experience (CURE)

“…Even with the small sample size after Round 2 ( n = 8), students were able to conclude that related samples 24–26 arising at this point suggested those might be highly functional satCs (students were asked to try to draw initial conclusions in their end‐of‐semester lab reports [see Supplemental Information for lab report guidelines]). Indeed, additional rounds of SELEX completed after the course ended revealed this to be the case 28 …”

“…Seniors graduated immediately following the conclusion of the spring semester in which they took the class so they were not able to continue the work, but of the 25 non‐seniors who took the course during one of the three offerings, 13 students inquired about joining the lab and 9 of them were able to work in the research lab for various lengths of time—from at least one summer or one semester, to more than 1 year—to continue with the project. All 9 contributed significantly to the work such that they were co‐authors on manuscripts that ultimately were published 28,31 …”

“…Indeed, additional rounds of SELEX completed after the course ended revealed this to be the case. 28…”

“…All 9 contributed significantly to the work such that they were coauthors on manuscripts that ultimately were published. 28,31 As an optional part of the attitudinal survey, many students wrote free-response comments stating that the semester-long research project was their most interesting, practical, and useful lab course of their college career (-Table S4; 28 of 43 students provided comments, and the entirety of all 28 comments are written in the table). For example, students commented that integration of methods into a project was relevant, they felt more prepared for future labwork, and that they enjoyed being able to perform novel research.…”

“… Turnip crinkle virus (TCV) and satellite C (satC) RNAs and their effect on plant leaves. (a) Current satC RNA structure model based off SHAPE and SELEX data acquired following the in‐class SELEX work 28,31 and drawn using RNA2Drawer 40 . (b) Top , schematic representation of TCV, a single‐strand positive‐sense RNA virus with a 4053 base genome that encodes 5 proteins.…”