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Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

DeOcesano-Pereira C, Amaral MS, Parreira KS, Ayupe AC, Jacysyn JF, Amarante-Mendes GP, Reis EM, Verjovski-Almeida S - Nucleic Acids Res. (2014)

Bottom Line: These effects were abrogated in the presence of INXS knockdown.Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis.BCL-XS protein accumulation was detected upon INXS overexpression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil.

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Characterization of INXS biogenesis, stability and cellular localization. (A) RNAPII inhibition with α-amanitin decreases the levels of INXS. Known RNAPII-transcribed (ACTB, MYC) and RNAPIII-transcribed genes (pre-tRNATyr, 7SK) were assayed as controls. (B) The presence of a 5′-end cap modification in INXS was determined by digestion with terminator 5′-phosphate-dependent exonuclease (5′Exo) in combination with tobacco acid pyrophosphatase (TAP), as indicated. TUBA1C tubulin gene was assayed as a control. (C) INXS decay rate in cells treated with actinomycin D, a transcription inhibitor. MYC was measured in parallel as a positive control of the decay assay. (D) Relative distribution of INXS in the nuclear and cytoplasmic fractions (N/C ratio). The nuclear-enriched MALAT1 lincRNA and the 45S rRNA were used as nuclear fraction controls and the 18S rRNA as a cytoplasmic fraction control. As an additional control, western blot of the protein extracts from the fractions was performed, detecting GAPDH only in the cytoplasmic fraction, and histone H3 only in the nuclear fraction. Further controls for cell fractionation are described in the Supplementary Data. *(P <0.05), **(P <0.01) and ***(P <0.001).
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Figure 3: Characterization of INXS biogenesis, stability and cellular localization. (A) RNAPII inhibition with α-amanitin decreases the levels of INXS. Known RNAPII-transcribed (ACTB, MYC) and RNAPIII-transcribed genes (pre-tRNATyr, 7SK) were assayed as controls. (B) The presence of a 5′-end cap modification in INXS was determined by digestion with terminator 5′-phosphate-dependent exonuclease (5′Exo) in combination with tobacco acid pyrophosphatase (TAP), as indicated. TUBA1C tubulin gene was assayed as a control. (C) INXS decay rate in cells treated with actinomycin D, a transcription inhibitor. MYC was measured in parallel as a positive control of the decay assay. (D) Relative distribution of INXS in the nuclear and cytoplasmic fractions (N/C ratio). The nuclear-enriched MALAT1 lincRNA and the 45S rRNA were used as nuclear fraction controls and the 18S rRNA as a cytoplasmic fraction control. As an additional control, western blot of the protein extracts from the fractions was performed, detecting GAPDH only in the cytoplasmic fraction, and histone H3 only in the nuclear fraction. Further controls for cell fractionation are described in the Supplementary Data. *(P <0.05), **(P <0.01) and ***(P <0.001).

Mentions: Inspection of the ChIP-seq data from the ENCODE project (42) indicated that the RNA polymerase II (RNAPII) chromatin mark was enriched at the putative transcription start site of INXS within the BCL-X genomic locus. To determine if INXS is transcribed by RNAPII, cells were treated with the RNAPII inhibitor α-amanitin; ∼90% reduction of INXS levels was observed (Figure 3A). In addition, we determined that the INXS lncRNA is modified by a methyl-guanosine cap, using the tobacco acid pyrophosphatase/5′exonuclease assay (Figure 3B). The half-life of INXS was determined to be ∼3 h (Figure 3C). For comparison, the measured half-life of MYC was 29 min, which is comparable with its half-life in the literature (43). Cell fractionation experiments revealed that INXS is predominantly enriched in the nucleus (Figure 3D).


Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

DeOcesano-Pereira C, Amaral MS, Parreira KS, Ayupe AC, Jacysyn JF, Amarante-Mendes GP, Reis EM, Verjovski-Almeida S - Nucleic Acids Res. (2014)

Characterization of INXS biogenesis, stability and cellular localization. (A) RNAPII inhibition with α-amanitin decreases the levels of INXS. Known RNAPII-transcribed (ACTB, MYC) and RNAPIII-transcribed genes (pre-tRNATyr, 7SK) were assayed as controls. (B) The presence of a 5′-end cap modification in INXS was determined by digestion with terminator 5′-phosphate-dependent exonuclease (5′Exo) in combination with tobacco acid pyrophosphatase (TAP), as indicated. TUBA1C tubulin gene was assayed as a control. (C) INXS decay rate in cells treated with actinomycin D, a transcription inhibitor. MYC was measured in parallel as a positive control of the decay assay. (D) Relative distribution of INXS in the nuclear and cytoplasmic fractions (N/C ratio). The nuclear-enriched MALAT1 lincRNA and the 45S rRNA were used as nuclear fraction controls and the 18S rRNA as a cytoplasmic fraction control. As an additional control, western blot of the protein extracts from the fractions was performed, detecting GAPDH only in the cytoplasmic fraction, and histone H3 only in the nuclear fraction. Further controls for cell fractionation are described in the Supplementary Data. *(P <0.05), **(P <0.01) and ***(P <0.001).
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Figure 3: Characterization of INXS biogenesis, stability and cellular localization. (A) RNAPII inhibition with α-amanitin decreases the levels of INXS. Known RNAPII-transcribed (ACTB, MYC) and RNAPIII-transcribed genes (pre-tRNATyr, 7SK) were assayed as controls. (B) The presence of a 5′-end cap modification in INXS was determined by digestion with terminator 5′-phosphate-dependent exonuclease (5′Exo) in combination with tobacco acid pyrophosphatase (TAP), as indicated. TUBA1C tubulin gene was assayed as a control. (C) INXS decay rate in cells treated with actinomycin D, a transcription inhibitor. MYC was measured in parallel as a positive control of the decay assay. (D) Relative distribution of INXS in the nuclear and cytoplasmic fractions (N/C ratio). The nuclear-enriched MALAT1 lincRNA and the 45S rRNA were used as nuclear fraction controls and the 18S rRNA as a cytoplasmic fraction control. As an additional control, western blot of the protein extracts from the fractions was performed, detecting GAPDH only in the cytoplasmic fraction, and histone H3 only in the nuclear fraction. Further controls for cell fractionation are described in the Supplementary Data. *(P <0.05), **(P <0.01) and ***(P <0.001).
Mentions: Inspection of the ChIP-seq data from the ENCODE project (42) indicated that the RNA polymerase II (RNAPII) chromatin mark was enriched at the putative transcription start site of INXS within the BCL-X genomic locus. To determine if INXS is transcribed by RNAPII, cells were treated with the RNAPII inhibitor α-amanitin; ∼90% reduction of INXS levels was observed (Figure 3A). In addition, we determined that the INXS lncRNA is modified by a methyl-guanosine cap, using the tobacco acid pyrophosphatase/5′exonuclease assay (Figure 3B). The half-life of INXS was determined to be ∼3 h (Figure 3C). For comparison, the measured half-life of MYC was 29 min, which is comparable with its half-life in the literature (43). Cell fractionation experiments revealed that INXS is predominantly enriched in the nucleus (Figure 3D).

Bottom Line: These effects were abrogated in the presence of INXS knockdown.Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis.BCL-XS protein accumulation was detected upon INXS overexpression.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-900 São Paulo, SP, Brasil.

Show MeSH
Related in: MedlinePlus