Expression, purification and evaluation of recombinant human Syntaxin 18 as an endoribonuclease.
Bibliographic record
Abstract
Regulation of mRNA decay is a major control point in gene expression. The processes and key players of mRNA decay in bacteria and yeast are relatively well established and characterized. In contrast, there are major gaps in our understanding of mRNA decay machineries in mammalian cells. Endonucleases appear to be key players in mammalian mRNA degradation, but the enzymes responsible are largely unknown. This is partly due to the fact that mRNA cleavage products are highly unstable and therefore difficult to detect. A novel mammalian endoribonuclease from rat liver that could cleave c-myc coding region determinant (CRD) RNA in vitro has recently been purified, and initial MALDI-MS data indicated that a candidate protein was Syntaxin 18 (Stx18), a soluble N-ethylmaleimide sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE)-family member. Further work with recombinant human Stx18 also indicated that Stx18 possessed the ability to cleave c-myc CRD RNA in vitro. The main objective of this thesis was to determine the endonucleolytic domain of Stx18 utilizing a deletion mapping approach, whereby truncated mutant forms of Stx18 would be generated, purified, refolded, and tested for endonucleolytic activity. Following this, the key catalytic residues of Stx18 were to be determined using the alanine-scanning approach. Unfortunately, throughout the course of this work, new evidence came to light that questions if Stx18 does in fact possess endoribonucleolytic activity, and thus the objective of this thesis shifted somewhat towards definitive determination of whether or not Stx18 is an endoribonuclease. Irrefutably conclusive evidence that Stx18 does not possess endonuclease activity was not forthcoming, but other evidence presented herein strongly suggests that it is a small, as-yet unidentified co-purified protein that is responsible for the endonucleolytic activity seen.--Pii-iii.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 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".