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A short RNA stem-loop is necessary and sufficient for repression of gene expression during early logarithmic phase in trypanosomes.

Fernández-Moya SM, Carrington M, Estévez AM - Nucleic Acids Res. (2014)

Bottom Line: We have compared the transcriptomes of cultured procyclic Trypanosoma brucei cells in early and late logarithmic phases and found that ∼200 mRNAs were differentially regulated.When placed in the 3'-UTR of an unregulated transcript, the cis-element is sufficient to confer regulation in response to purines.To our knowledge, this is the first example of a discrete RNA element that can autonomously regulate gene expression in trypanosomes in response to an external factor and reveals an unprecedented purine-dependent signaling pathway that controls gene expression in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Parasitología y Biomedicina 'López-Neyra', IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento, s/n, 18016 Armilla, Granada, Spain.

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Effect of the stem–loop element on gene expression in different gene contexts. (A) Cell lines were generated which expressed luciferase under the control of actin (ACT) or mitochondrial malate dehydrogenase (mMDH) 3′-UTRs in which the regulatory stem–loop was inserted. (B) Effect of guanosine withdrawal in early log cultures expressing luciferase under the control of 3′-UTRs containing or lacking the regulatory stem–loop. The actual values of relative luciferase activity are indicated above each bar for convenience. (C) Sequences of the short (35 mer) wild-type stem–loop and mutated versions thereof. Mutations in the stem are indicated by asterisks. Detailed sequences and structures can be seen in Supplementary Figures S3 and S4. (D) Effect of shorter and mutant stem–loop versions on luciferase expression. Luciferase activity was normalized to that of a cell line expressing the reporter under the control of EP1 5′-UTR and actin 3-UTR (dashed line).
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Figure 7: Effect of the stem–loop element on gene expression in different gene contexts. (A) Cell lines were generated which expressed luciferase under the control of actin (ACT) or mitochondrial malate dehydrogenase (mMDH) 3′-UTRs in which the regulatory stem–loop was inserted. (B) Effect of guanosine withdrawal in early log cultures expressing luciferase under the control of 3′-UTRs containing or lacking the regulatory stem–loop. The actual values of relative luciferase activity are indicated above each bar for convenience. (C) Sequences of the short (35 mer) wild-type stem–loop and mutated versions thereof. Mutations in the stem are indicated by asterisks. Detailed sequences and structures can be seen in Supplementary Figures S3 and S4. (D) Effect of shorter and mutant stem–loop versions on luciferase expression. Luciferase activity was normalized to that of a cell line expressing the reporter under the control of EP1 5′-UTR and actin 3-UTR (dashed line).

Mentions: To study whether the RNA element was sufficient to provide regulation in a different gene context, we modified the luciferase transgene construct to insert the stem–loop 149 nucleotides downstream the luciferase stop codon. As shown in Figure 7A, luciferase activity decreased ∼10-fold in early log phase when the regulatory element was placed into the wild-type actin 3′-UTR. In a second experiment, the stem–loop was inserted within a 3′-UTR derived from the mitochondrial malate dehydrogenase (mMDH) gene, which was not normally regulated during growth cycle (Supplementary Table S1). Cells expressing luciferase fused to wild-type mMDH 3′-UTR showed an activity close to that observed in the actin control cell line (Figure 7A). However, the insertion of the stem–loop within the 3′-UTR of mMDH resulted in a 20-fold decrease in luciferase activity in early log phase.


A short RNA stem-loop is necessary and sufficient for repression of gene expression during early logarithmic phase in trypanosomes.

Fernández-Moya SM, Carrington M, Estévez AM - Nucleic Acids Res. (2014)

Effect of the stem–loop element on gene expression in different gene contexts. (A) Cell lines were generated which expressed luciferase under the control of actin (ACT) or mitochondrial malate dehydrogenase (mMDH) 3′-UTRs in which the regulatory stem–loop was inserted. (B) Effect of guanosine withdrawal in early log cultures expressing luciferase under the control of 3′-UTRs containing or lacking the regulatory stem–loop. The actual values of relative luciferase activity are indicated above each bar for convenience. (C) Sequences of the short (35 mer) wild-type stem–loop and mutated versions thereof. Mutations in the stem are indicated by asterisks. Detailed sequences and structures can be seen in Supplementary Figures S3 and S4. (D) Effect of shorter and mutant stem–loop versions on luciferase expression. Luciferase activity was normalized to that of a cell line expressing the reporter under the control of EP1 5′-UTR and actin 3-UTR (dashed line).
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Figure 7: Effect of the stem–loop element on gene expression in different gene contexts. (A) Cell lines were generated which expressed luciferase under the control of actin (ACT) or mitochondrial malate dehydrogenase (mMDH) 3′-UTRs in which the regulatory stem–loop was inserted. (B) Effect of guanosine withdrawal in early log cultures expressing luciferase under the control of 3′-UTRs containing or lacking the regulatory stem–loop. The actual values of relative luciferase activity are indicated above each bar for convenience. (C) Sequences of the short (35 mer) wild-type stem–loop and mutated versions thereof. Mutations in the stem are indicated by asterisks. Detailed sequences and structures can be seen in Supplementary Figures S3 and S4. (D) Effect of shorter and mutant stem–loop versions on luciferase expression. Luciferase activity was normalized to that of a cell line expressing the reporter under the control of EP1 5′-UTR and actin 3-UTR (dashed line).
Mentions: To study whether the RNA element was sufficient to provide regulation in a different gene context, we modified the luciferase transgene construct to insert the stem–loop 149 nucleotides downstream the luciferase stop codon. As shown in Figure 7A, luciferase activity decreased ∼10-fold in early log phase when the regulatory element was placed into the wild-type actin 3′-UTR. In a second experiment, the stem–loop was inserted within a 3′-UTR derived from the mitochondrial malate dehydrogenase (mMDH) gene, which was not normally regulated during growth cycle (Supplementary Table S1). Cells expressing luciferase fused to wild-type mMDH 3′-UTR showed an activity close to that observed in the actin control cell line (Figure 7A). However, the insertion of the stem–loop within the 3′-UTR of mMDH resulted in a 20-fold decrease in luciferase activity in early log phase.

Bottom Line: We have compared the transcriptomes of cultured procyclic Trypanosoma brucei cells in early and late logarithmic phases and found that ∼200 mRNAs were differentially regulated.When placed in the 3'-UTR of an unregulated transcript, the cis-element is sufficient to confer regulation in response to purines.To our knowledge, this is the first example of a discrete RNA element that can autonomously regulate gene expression in trypanosomes in response to an external factor and reveals an unprecedented purine-dependent signaling pathway that controls gene expression in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Parasitología y Biomedicina 'López-Neyra', IPBLN-CSIC, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento, s/n, 18016 Armilla, Granada, Spain.

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