Methods for isolation of messenger RNA from biological samples

Cheng, Mingyu; Tao, Wan-Bing; Yuan, Bi-Feng

doi:10.1039/d0ay01912g

Cited by 7 publications

(5 citation statements)

References 60 publications

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“…31,58 The inability to confidently obtain highly pure mRNA samples has limited the utility of nucleoside LC-MS/MS for studying these molecules. Recently, small RNA depletion steps have begun to be incorporated into mRNA purification pipelines to remove residual tRNA contamination; 59 however, the highest efficiency purifications typically require expensive instrumentation and materials (liquid chromatograph and size exclusion column) 33 or expertise in RNA gel purification. 32 Despite these improvements, most reports do not provide the extensive mRNA purity quality controls necessary to confidently confirm the removal of ncRNA from mRNA samples.…”

Section: Development Of Highly Sensitive Lc-ms/ms Methods For Simulta...mentioning

confidence: 99%

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

et al. 2023

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Section: Development Of Highly Sensitive Lc-ms/ms Methods For Simulta...mentioning

confidence: 99%

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

et al. 2023

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“…The inability to obtain convincingly pure mRNA samples has long limited the utility of LC-MS/MS for studying these molecules. Recently, small RNA depletion steps have begun to be incorporated into mRNA purification pipelines to remove residual tRNA contamination 80 ; however, the highest efficiency purifications typically require expensive instrumentation and materials (liquid chromatograph and size exclusion column) 23 or expertise in RNA gel purification 21 . Despite these improvements, most reports do not provide adequate mRNA purity quality control to confirm removal of ncRNA for confident mRNA modification analyses.…”

Section: Resultsmentioning

confidence: 99%

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Jones

Franco

Smith

et al. 2022

Preprint

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Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

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“…Isolating mRNA is technically challenging owing to its susceptibility to degradation by many RNA-degrading enzymes and buffers [10]. This fact makes the ability to deliver an mRNA vaccine in a lipid nanoparticle vesicle through a needle into different cell types a remarkable success.…”

Section: Discussionmentioning

confidence: 99%

In vitro Characterization of SARS-CoV-2 Protein Translated from the Moderna mRNA-1273 Vaccine

Veenstra

Injeti

Pauley

2022

Preprint

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Extensive research around mRNA vaccines and their proposed utility during the current COVID-19 pandemic resulted in many publications concerning the SARS-Cov-2 spike protein and angiotensin converting enzyme-2 receptor-binding domain of the virus, but none describe the characteristics of the full-length protein obtained from the modified/synthetic mRNA that is part of the Moderna and Pfizer-BioNTech vaccines. In this paper, we provide the first data characterizing the actual proteins produced by mouse and human cells in culture that had been incubated up to 30 minutes with the commercial vaccine produced by Moderna (i.e., Spikevax). The mRNA vaccine continues to produce proteins up to 12-14 days after introduction to the cells. The molecular weight of the SARS-CoV-2 encoded protein ranges from 135-200 kilodaltons depending on the extent of glycosylation.

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Methods for isolation of messenger RNA from biological samples

Abstract: RNA molecules contain many chemical modifications that can regulate a variety of biological processes. Messenger RNA (mRNA) molecules are the critical components in the central dogma of molecular biology. The...

Cited by 7 publications

References 60 publications

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

In vitro Characterization of SARS-CoV-2 Protein Translated from the Moderna mRNA-1273 Vaccine

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