Deciphering messenger ribonucleoprotein composition using single molecule resolution microscopy
Bibliographic record
Abstract
Messenger RNA (mRNA) molecules expressed in the nuclei of eukaryotic organisms associate with many proteins to facilitate processing and maturation of mRNA, as well as packaging into messenger ribonucleoprotein particle (mRNP) complexes to facilitate nuclear export of transcripts.Existing methods used to study mRNPs have succeeded in identifying a number of proteins involved in mRNP biogenesis, but have failed to provide information with regards to mRNP structure or the heterogeneous pool of mRNPs existing in the cell.Here we describe steps taken towards the development of a novel technique aimed at quantifying protein abundance, in terms of stoichiometry, of mRNP proteins on specifically purified mRNA transcripts from cellular extracts by affinity purification and single-molecule microscopy.In developing this method, we aim to investigate the influence of mRNA length on mRNP protein stoichiometry, and to determine how mRNAs of different lengths may use the pool of mRNP biogenesis factors differently to achieve effective nuclear export. Résumé:Les molécules d'ARN messager (ARNm) exprimées dans les noyaux des organismes eucaryotes s'associent à certaines protéines pour faciliter le traitement et la maturation de l'ARNm, ainsi que pour former des complexes de particules ribonucléoprotéines messagères (RNPm) pour faciliter l'exportation hors du noyau.Les méthodes existantes utilisées pour étudier les RNPm ont réussi à identifier un certain nombre de protéines impliquées dans la biogenèse de la RNPm, mais ne parviennent pas à fournir des informations sur la structure de la RNPm ou l'hétérogénéité des RNPm qui existent dans la cellule.Nous décrivons ici les mesures prises pour le développement d'une nouvelle technique visant à quantifier l'abondance des protéines, en termes de stœchiométrie, des RNPm sur des ARNm spécifiquement purifiés à partir d'extraits cellulaires par purification d'affinité et microscopie à molécule unique.En développant cette méthode, nous cherchons à étudier l'influence de la longueur de l'ARNm sur la stœchiométrie des RNPm, afin de voir comment les ARNm de différentes longueurs peuvent utiliser différemment les facteurs de biogenèse de la RNPm pour faciliter l'exportation hors du noyau.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.001 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".