Neighboring inteins interfere with one another's homing capacity
Israela Turgeman-Grott1, Danielle Arsenault2, Dekel Yahav1
1The Shmunis School of Biomedicine and Cancer Research, Faculty of Life Sciences, Tel Aviv University, P.O. Box 39040, 6997801 Tel Aviv, Israel.
Abstract:
Inteins are mobile genetic elements that invade conserved genes across all domains of life and viruses. In some instances, a single gene will have several intein insertion sites. In Haloarchaea, the minichromosome maintenance (MCM) protein at the core of replicative DNA helicase contains four intein insertion sites within close proximity, where two of these sites (MCM-a and MCM-d) are more likely to be invaded. A haloarchaeon that harbors both MCM-a and MCM-d inteins, Haloferax mediterranei, was studied in vivo to determine intein invasion dynamics and the interactions between neighboring inteins. Additionally, invasion frequencies and the conservation of insertion site sequences in 129 Haloferacales mcm homologs were analyzed to assess intein distribution across the order. We show that the inteins at MCM-a and MCM-d recognize and cleave their respective target sites and, in the event that only one empty intein invasion site is present, readily initiate homing (i.e. single homing). However, when two inteins are present co-homing into an intein-free target sequence is much less effective. The two inteins are more effective when invading alleles that already contain an intein at one of the two sites. Our in vivo and computational studies also support that having a proline in place of a serine as the first C-terminal extein residue of the MCM-d insertion site prevents successful intein splicing, but does not stop recognition of the insertion site by the intein's homing endonuclease.
Insights
Inteins are mobile genetic elements. In Haloarchaea, the minichromosome maintenance (MCM) protein
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Inteins are mobile genetic elements found across life, capable of invading host genes.
- The minichromosome maintenance (MCM) protein, crucial for DNA replication, presents multiple intein insertion sites in Haloarchaea.
- Specific intein insertion sites (MCM-a and MCM-d) are frequently targeted within the MCM protein.
Purpose of the Study:
- To investigate intein invasion dynamics and interactions between adjacent inteins in the MCM protein of *Haloferax mediterranei*.
- To analyze the frequency and conservation of intein insertion sites in MCM homologs across the Haloferacales order.
- To understand the mechanisms governing intein homing and splicing in haloarchaeal MCM proteins.
Main Methods:
- In vivo studies in *Haloferax mediterranei* to observe intein invasion and splicing.
- Computational analysis of MCM homologs from 129 Haloferacales species.
- Assessing intein recognition, cleavage, and homing efficiency under different invasion scenarios.
Main Results:
- Inteins at MCM-a and MCM-d sites are recognized and cleave their targets.
- Single intein homing is efficient, but co-homing into intein-free sites is less effective.
- Invasion is more successful when one intein is already present at a neighboring site.
- A specific amino acid substitution (Proline for Serine) at the MCM-d site prevents splicing but not recognition.
Conclusions:
- Intein invasion and homing in haloarchaeal MCM proteins exhibit complex dynamics, influenced by the presence of neighboring inteins.
- The efficiency of intein homing is context-dependent, favoring invasion of already partially invaded alleles.
- Specific extein residues can modulate intein splicing without abolishing intein recognition, highlighting intricate host-intein interactions.
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