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THOC5 controls 3'end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100).

Tran DD, Saran S, Williamson AJ, Pierce A, Dittrich-Breiholz O, Wiehlmann L, Koch A, Whetton AD, Tamura T - Nucleic Acids Res. (2014)

Bottom Line: Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun.THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes.These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.

View Article: PubMed Central - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.

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Most IEGs were THOC5 dependent, however the redundant backup pathways partially rescue their upregulation. (A) ERT2Cre THOC5 (flox/flox) MEF cells were treated as described in Figure 2A and semi-quantitative RT-PCR were performed as indicated. Primers were located at different exons (Table 1). (B) Protein and total RNAs were isolated from nuclear and cytoplasmic fractions after 1 or 2 h serum stimulation. Proteins were used for THOC5, Histone H3 or GAPDH-specific immunoblot (Blot) and RNAs were used for RT-PCR as indicated. Three independent experiments were performed.
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Figure 3: Most IEGs were THOC5 dependent, however the redundant backup pathways partially rescue their upregulation. (A) ERT2Cre THOC5 (flox/flox) MEF cells were treated as described in Figure 2A and semi-quantitative RT-PCR were performed as indicated. Primers were located at different exons (Table 1). (B) Protein and total RNAs were isolated from nuclear and cytoplasmic fractions after 1 or 2 h serum stimulation. Proteins were used for THOC5, Histone H3 or GAPDH-specific immunoblot (Blot) and RNAs were used for RT-PCR as indicated. Three independent experiments were performed.

Mentions: We next investigated the IEGs, whose upregulation was reduced in the absence of THOC5. We selected 25 genes from those that displayed a >50% reduction in gene induction upon depletion of THOC5 (Table 2, Group [c]). To eliminate genes which are increased in expression indirectly, ERT2Cre THOC5 (flox/flox) MEF cells were treated with and without cycloheximide then stimulated with serum. Sixteen genes were still enhanced in expression in the presence of cycloheximide (Supplementary Figure S1). We further analyzed kinetics of upregulation of 10 genes, namely Id1, Id3, Wnt11 (Figure 2), Dusp2, Errfi1, Hes1, Lif, Myc, Smad7 and Snai1 (Figure 3) in the presence or absence of THOC5. Quantitative RT-PCR was performed using primers that locate at different exons (Table 1). Among these genes, Id1, Id3 and Wnt11 mRNAs were not increased in expression at any time points in THOC5-depleted cells (Figure 2A and B), indicating that THOC5 is indispensable for the processing of these mRNAs. Katahira etal. (14) recently showed that THOC5 plays a role in 3′end-processing of THOC5 target genes in HeLa cells. We next examined increased expression of uncleaved forms of Id1, Id3 and Wnt11 mRNAs upon serum stimulation. Primer pairs were designed that locate up- and down-stream of cleavage sites based on the NCBI data base (Table 1). As shown in Figure 2C, upon depletion of THOC5, the uncleaved Id1, Id3 and Wnt11 mRNAs were upregulated to an even greater level than they were in the presence of THOC5. To confirm these data, we next isolated chromatin associated mRNAs (Figure 2E) and semi-quantitative RT-PCR was performed. In the absence of THOC5, chromatin associated Id1 and Id3 mRNAs accumulated within 30 min (Figure 2F). Uncleaved and chromatin associated Id3 mRNA was increased more than 2-fold in the absence of THOC5 within 30 min (Figure 2D and G, qRT-PCR), suggesting that these genes were transcribed, but that the mRNA was not cleaved in the absence of THOC5.


THOC5 controls 3'end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100).

Tran DD, Saran S, Williamson AJ, Pierce A, Dittrich-Breiholz O, Wiehlmann L, Koch A, Whetton AD, Tamura T - Nucleic Acids Res. (2014)

Most IEGs were THOC5 dependent, however the redundant backup pathways partially rescue their upregulation. (A) ERT2Cre THOC5 (flox/flox) MEF cells were treated as described in Figure 2A and semi-quantitative RT-PCR were performed as indicated. Primers were located at different exons (Table 1). (B) Protein and total RNAs were isolated from nuclear and cytoplasmic fractions after 1 or 2 h serum stimulation. Proteins were used for THOC5, Histone H3 or GAPDH-specific immunoblot (Blot) and RNAs were used for RT-PCR as indicated. Three independent experiments were performed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4231767&req=5

Figure 3: Most IEGs were THOC5 dependent, however the redundant backup pathways partially rescue their upregulation. (A) ERT2Cre THOC5 (flox/flox) MEF cells were treated as described in Figure 2A and semi-quantitative RT-PCR were performed as indicated. Primers were located at different exons (Table 1). (B) Protein and total RNAs were isolated from nuclear and cytoplasmic fractions after 1 or 2 h serum stimulation. Proteins were used for THOC5, Histone H3 or GAPDH-specific immunoblot (Blot) and RNAs were used for RT-PCR as indicated. Three independent experiments were performed.
Mentions: We next investigated the IEGs, whose upregulation was reduced in the absence of THOC5. We selected 25 genes from those that displayed a >50% reduction in gene induction upon depletion of THOC5 (Table 2, Group [c]). To eliminate genes which are increased in expression indirectly, ERT2Cre THOC5 (flox/flox) MEF cells were treated with and without cycloheximide then stimulated with serum. Sixteen genes were still enhanced in expression in the presence of cycloheximide (Supplementary Figure S1). We further analyzed kinetics of upregulation of 10 genes, namely Id1, Id3, Wnt11 (Figure 2), Dusp2, Errfi1, Hes1, Lif, Myc, Smad7 and Snai1 (Figure 3) in the presence or absence of THOC5. Quantitative RT-PCR was performed using primers that locate at different exons (Table 1). Among these genes, Id1, Id3 and Wnt11 mRNAs were not increased in expression at any time points in THOC5-depleted cells (Figure 2A and B), indicating that THOC5 is indispensable for the processing of these mRNAs. Katahira etal. (14) recently showed that THOC5 plays a role in 3′end-processing of THOC5 target genes in HeLa cells. We next examined increased expression of uncleaved forms of Id1, Id3 and Wnt11 mRNAs upon serum stimulation. Primer pairs were designed that locate up- and down-stream of cleavage sites based on the NCBI data base (Table 1). As shown in Figure 2C, upon depletion of THOC5, the uncleaved Id1, Id3 and Wnt11 mRNAs were upregulated to an even greater level than they were in the presence of THOC5. To confirm these data, we next isolated chromatin associated mRNAs (Figure 2E) and semi-quantitative RT-PCR was performed. In the absence of THOC5, chromatin associated Id1 and Id3 mRNAs accumulated within 30 min (Figure 2F). Uncleaved and chromatin associated Id3 mRNA was increased more than 2-fold in the absence of THOC5 within 30 min (Figure 2D and G, qRT-PCR), suggesting that these genes were transcribed, but that the mRNA was not cleaved in the absence of THOC5.

Bottom Line: Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun.THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes.These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing.

View Article: PubMed Central - PubMed

Affiliation: Institut fuer Biochemie, OE4310, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany.

Show MeSH