Identifying novel splicing factors of the group II intron L1.LtrB from «Lactococcus lactis»
Bibliographic record
Abstract
Group II (GII) introns are large ribozymes that can autocatalytically excise themselves from pre-mRNA transcripts. They are found in bacteria, archaea, and the organelles of lower eukaryotes and land plants. GII introns contain all the necessary components for splicing, but protein splicing factors are essential to induce proper tridimensional folding of these intron RNAs in vivo. GII introns in bacteria and archaea usually encode their own splicing factor, termed the intron-encoded protein (IEP). In contrast, most GII introns in land plants do not encode an IEP. Instead, such GII introns depend on a diverse set of RNA-binding proteins to act as splicing factors. We aim to explore the potential diversity of group II intron splicing factors in bacteria.We study the model group II intron Ll.LtrB, from the Gram-positive bacterium Lactococcus lactis. Ll.LtrB encodes the IEP LtrA, which is essential for efficient and accurate splicing. Ll.LtrB interrupts the gene coding for the relaxase enzyme (LtrB), necessary to initiate the transfer of conjugative elements in L. lactis. Since ligation of ltrB exons is necessary for conjugation, splicing efficiency of Ll.LtrB from the ltrB pre-mRNA controls the transfer efficiency of conjugative elements. We took advantage of this relationship to develop a sensitive conjugation assay to study a splicing-deficient mutant of Ll.LtrB, which lacks LtrA (Ll.LtrBΔLtrA). Previously, a genomic expression library of L. lactis was generated in a conjugative plasmid. A conjugation assay was performed and led to the selection of three plasmids containing genomic fragments (Lib7,9,11) that can rescue conjugation efficiency in the absence of LtrA (Ll.LtrBΔLtrA).Lib11 encodes five putative open reading frames (ORFs) and two partial ORFs. We used two different strategies to determine which ORFs may be responsible for the observed increase in conjugation efficiency, and hence splicing rescue. First, we expressed individual ORFs from a conjugative plasmid. Second, we knocked out expression of individual ORFs within the original Lib11 construct. From these strategies, ORF1 was determined to increase conjugation efficiency, but the effect was variable. Similarly, further studies revealed that Lib11-containing strains also displayed variable levels of conjugation. We thus performed a series of control conjugations and identified conditions in which the variability in conjugation efficiency is reduced. We can now identify which of the ORFs is responsible for the increase in conjugation efficiency.
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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 | 0.000 | 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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".