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Ribonuclease III-mediated processing of specific Neisseria meningitidis mRNAs
Eliana De Gregorio1, Chiara Abrescia, M Stella Carlomagno
1Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università degli Studi di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, Italy.
Abstract:
Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly.
Insights
Neisseria meningitidis Correia (nemis) elements are processed by RNase III, but specific terminal repeat sequences can inhibit this cleavage. This regulation impacts mRNA levels and stability.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Neisseria meningitidis possesses small DNA insertion sequences called nemis elements, comprising ~2% of its genome.
- These nemis elements feature terminal inverted repeats (TIRs) of 26-27 base pairs.
- Nemis elements are co-transcribed with flanking genes, and resulting mRNAs are processed at double-stranded RNA structures formed by TIRs.
Purpose of the Study:
- To investigate the processing of nemis+ RNAs by N. meningitidis RNase III.
- To determine the role of TIR sequence complementarity in RNA hairpin processing.
- To understand how nemis element processing affects mRNA levels and stability.
Main Methods:
- In vitro cleavage assays using N. meningitidis RNase III.
- Electrophoretic mobility shift assays (EMSA) to study protein-RNA interactions.
- Analysis of mRNA levels and RNA stability in vivo and in vitro.
Main Results:
- N. meningitidis RNase III efficiently cleaves RNA hairpins formed by nemis elements with identical TIRs (26/26 and 27/27 bp).
- Hairpins formed by mismatched TIRs (26/27 bp) inhibit RNase III cleavage both in vitro and in vivo.
- All hairpin types formed similar complexes with RNase III in EMSA, suggesting binding is not the sole determinant of cleavage.
- Variations in nemis+ and nemis- mRNA levels and in vitro RNA segment stability were observed.
Conclusions:
- RNase III-mediated processing of nemis+ RNAs is sequence-dependent, regulated by TIR complementarity.
- Mismatched TIRs in nemis elements can inhibit processing, potentially influencing gene expression.
- The findings highlight a novel regulatory mechanism impacting mRNA processing and stability in N. meningitidis.