Lysate and cell-based assays to probe the translational role of RNA helicases

Wilkins, Kevin; Venkataramanan, Srivats; Floor, Stephen N.

doi:10.1016/bs.mie.2022.03.032

Cited by 3 publications

(8 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ribosome profiling in prior work showed that the loss of DDX3X caused a decrease in ribosome density on a subset of transcripts, including ODC1, RAC1, and HMBS ( Calviello et al 2021 ). The 5′ UTRs of these transcripts require DDX3X for efficient translation in in vitro and in cell assays ( Calviello et al 2021 ; Wilkins et al 2022 ). We define these sequences as DDX3X-sensitive 5′ UTRs and reasoned we could use them as the foundation of a reporter for DDX3X translation activity in cells ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel reporter for helicase activity in translation uncovers DDX3X interactions

Wilkins,

Schroeder,

et al. 2024

RNA

Self Cite

View full text Add to dashboard Cite

DDX3X regulates the translation of a subset of human transcripts containing complex 5′ untranslated regions (5′ UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5′ UTRs to measure DDX3X mediated translational activity in cells. To directly measure RNA structure in DDX3X dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5′ UTRs and then employed HART to investigate how sequence alterations influence DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X’s interaction with the translational machinery. HART revealed that both DDX3X’s association with the translational machinery as well as its helicase activity are required for its function in promoting the translation of DDX3X sensitive 5′ UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally-encoded, colorimetric measurement of DDX3X-dependent translation in cells.

show abstract

Section: Resultsmentioning

confidence: 99%

A novel reporter for helicase activity in translation uncovers DDX3X interactions

Wilkins,

Schroeder,

et al. 2024

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…Ribosome profiling in prior work showed that the loss of DDX3X caused a decrease in ribosome density on a subset of transcripts, including ODC1, RAC1, and HMBS 19 . The 5′ untranslated regions (5′ UTR) of these transcripts requires DDX3X for efficient translation in in vitro and in cell assays 19,21 . We define these sequences as DDX3X-sensitive 5′ UTRs and reasoned we could use them as the foundation of a reporter for DDX3X translation activity in cells (Figure 1A, B).…”

Section: Resultsmentioning

confidence: 99%

“…First, we in vitro transcribed RNA as described 21 . RAC1 and ODC1 5′ UTRs were placed under control of a T7 promoter and in front of luciferase ORF and transcribed using T7 polymerase 19,21 . For Shape-MaP, 1000 ng of the transcribed RNA in 12 μL water was denatured at 95 °C for 2 min and then snap cooled on ice for 2 min.…”

Section: Shape-mapmentioning

confidence: 99%

“…To measure RNA structures using SHAPE-MaP, mRNAs containing the RAC1 and ODC1 5′ UTRs and the luciferase ORF were in vitro transcribed using T7 polymerase 19,21 . RNA was then folded, probed with NAI or DMSO as control, and then reverse transcribed and sequenced.…”

Section: Structural Probing Of the Rac1 And Odc1 5′ Utrsmentioning

confidence: 99%

“…SHAPE-MaP was conducted as described 30,31 . First, we in vitro transcribed RNA as described 21 . RAC1 and ODC1 5′ UTRs were placed under control of a T7 promoter and in front of luciferase ORF and transcribed using T7 polymerase 19,21 .…”

Section: Shape-mapmentioning

confidence: 99%

See 2 more Smart Citations

Determinants of DDX3X sensitivity uncovered using a helicase activity in translation reporter

Wilkins,

Schroeder,

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

DDX3X regulates the translation of a subset of human transcripts containing complex 5′ untranslated regions (5′ UTRs). In this study we developed the helicase activity reporter for translation (HART) which uses DDX3X-sensitive 5′ UTRs to measure DDX3X mediated translational activity in cells. To dissect the structural underpinnings of DDX3X dependent translation, we first used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5′ UTRs and then employed HART to investigate how their perturbation impacts DDX3X-sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3Xs interaction with the translational machinery. HART revealed that both DDX3Xs association with the ribosome complex as well as its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5′ UTRs. These findings suggest DDX3X plays a crucial role regulating translation through its interaction with the translational machinery during ribosome scanning, and establish the HART reporter as a robust, lentivirally encoded measurement of DDX3X-dependent translation in cells.

show abstract

Production and secretion of recombinant spider silk in Bacillus megaterium

Connor,

Zha,

Koffas

2024

Microb Cell Fact

View full text Add to dashboard Cite

Background Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. Results In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. Conclusion It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

show abstract

Lysate and cell-based assays to probe the translational role of RNA helicases

Cited by 3 publications

References 39 publications

A novel reporter for helicase activity in translation uncovers DDX3X interactions

A novel reporter for helicase activity in translation uncovers DDX3X interactions

Determinants of DDX3X sensitivity uncovered using a helicase activity in translation reporter

Production and secretion of recombinant spider silk in Bacillus megaterium

Contact Info

Product

Resources

About