Limits...
Characterizing TDP-43 interaction with its RNA targets.

Bhardwaj A, Myers MP, Buratti E, Baralle FE - Nucleic Acids Res. (2013)

Bottom Line: Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself.In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop.In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

ABSTRACT
One of the most important functional features of nuclear factor TDP-43 is its ability to bind UG-repeats with high efficiency. Several cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation-sequencing (RIP-seq) analyses have indicated that TDP-43 in vivo can also specifically bind loosely conserved UG/GU-rich repeats interspersed by other nucleotides. These sequences are predominantly localized within long introns and in the 3'UTR of various genes. Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself. In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop. In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches. We show that the binding affinity to the UG-like sequences has a dissociation constant (Kd) of ∼110 nM compared with a Kd of 8 nM for straight UGs, and have mapped the region of contact between protein and RNA. In addition, our results indicate that the local concentration of UG dinucleotides in the CLIP sequences is one of the major factors influencing the interaction of these RNA sequences with TDP-43.

Show MeSH

Related in: MedlinePlus

Titration experiments for various TDP–RNA complexes using circular dichroism. Starting from 0 μM (blue), 5 μM (violet), 10 μM (green) and 15 μM (red) of GST-TDP (101–261) were used to perform titration experiments against a fixed concentration of various RNAs. Panels A, B, C and D shows the subtracted CD spectra of (UG)6 (18 μM), CLIP34nt (10 μM), CLIP6 (9 μM) and CLIP34nt_UG6 (8 μM) in the presence of various concentration of GST-TDP (101–261), respectively. Panel E and F show the subtracted CD spectra of CLIP34nt_UG6 (8 μM) in the presence of various concentrations of GST-TDPmut2 (101–261) and GST-TDPmut1 (101–261), respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643599&req=5

gkt189-F4: Titration experiments for various TDP–RNA complexes using circular dichroism. Starting from 0 μM (blue), 5 μM (violet), 10 μM (green) and 15 μM (red) of GST-TDP (101–261) were used to perform titration experiments against a fixed concentration of various RNAs. Panels A, B, C and D shows the subtracted CD spectra of (UG)6 (18 μM), CLIP34nt (10 μM), CLIP6 (9 μM) and CLIP34nt_UG6 (8 μM) in the presence of various concentration of GST-TDP (101–261), respectively. Panel E and F show the subtracted CD spectra of CLIP34nt_UG6 (8 μM) in the presence of various concentrations of GST-TDPmut2 (101–261) and GST-TDPmut1 (101–261), respectively.

Mentions: To further explore the conformational changes in RNA due to protein binding, titration experiments were then performed in which a fixed concentration of all the four RNA samples [CLIP34nt, CLIP6, CLIP34nt_UG6 and (UG)6] were titrated against various concentration of GST-TDP (101–261) (Figure 4). In case of (UG)6 RNA, the peaks (at 275 and 200 nm) were found to deviate from the typical A-type helix spectrum and were of less intensity, presumably owing to its weak tendency towards proper base stacking. A clear reduction in the peak intensities (at 200 and 275 nm) was observed for (UG)6 RNA with increasing concentrations of GST-TDP (101–261), and this could be interpreted as a change in RNA conformation due to TDP-43 binding (Figure 4A). Compared with this situation, the subtracted CD spectra of CLIP34nt displayed subtle changes at 265 nm, whereas the subtracted spectra of CLIP6 RNA showed that the negative peak at 210 nm (in unbound state) shifted to 200 nm on binding with protein, but the peak at 268 nm remained unchanged. These subtle changes may be because of the presence of comparatively weak interaction of CLIP34nt and CLIP6 with GST-TDP (101–261) with respect to (UG)6 RNA (Figure 4B and C). Finally, in the case of the CLIP34nt_UG6 RNA sample, it was observed that the intensity at 255 nm decreased with increasing GST-TDP (101–261) concentrations like it was observed with (UG)6 RNA (Figure 4D). The CD titration experiment was also performed with GST-TDPmut2 using CLIP34nt_UG6 RNA, and this mutant displayed the same binding affinity as GST-TDP (101–261) to all the target RNA oligos. The substracted spectra displayed the same pattern like the one observed for GST-TDP (101–261), with just some subtle difference at 15 μM protein concentration (Figure 4E).Figure 4.


Characterizing TDP-43 interaction with its RNA targets.

Bhardwaj A, Myers MP, Buratti E, Baralle FE - Nucleic Acids Res. (2013)

Titration experiments for various TDP–RNA complexes using circular dichroism. Starting from 0 μM (blue), 5 μM (violet), 10 μM (green) and 15 μM (red) of GST-TDP (101–261) were used to perform titration experiments against a fixed concentration of various RNAs. Panels A, B, C and D shows the subtracted CD spectra of (UG)6 (18 μM), CLIP34nt (10 μM), CLIP6 (9 μM) and CLIP34nt_UG6 (8 μM) in the presence of various concentration of GST-TDP (101–261), respectively. Panel E and F show the subtracted CD spectra of CLIP34nt_UG6 (8 μM) in the presence of various concentrations of GST-TDPmut2 (101–261) and GST-TDPmut1 (101–261), respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643599&req=5

gkt189-F4: Titration experiments for various TDP–RNA complexes using circular dichroism. Starting from 0 μM (blue), 5 μM (violet), 10 μM (green) and 15 μM (red) of GST-TDP (101–261) were used to perform titration experiments against a fixed concentration of various RNAs. Panels A, B, C and D shows the subtracted CD spectra of (UG)6 (18 μM), CLIP34nt (10 μM), CLIP6 (9 μM) and CLIP34nt_UG6 (8 μM) in the presence of various concentration of GST-TDP (101–261), respectively. Panel E and F show the subtracted CD spectra of CLIP34nt_UG6 (8 μM) in the presence of various concentrations of GST-TDPmut2 (101–261) and GST-TDPmut1 (101–261), respectively.
Mentions: To further explore the conformational changes in RNA due to protein binding, titration experiments were then performed in which a fixed concentration of all the four RNA samples [CLIP34nt, CLIP6, CLIP34nt_UG6 and (UG)6] were titrated against various concentration of GST-TDP (101–261) (Figure 4). In case of (UG)6 RNA, the peaks (at 275 and 200 nm) were found to deviate from the typical A-type helix spectrum and were of less intensity, presumably owing to its weak tendency towards proper base stacking. A clear reduction in the peak intensities (at 200 and 275 nm) was observed for (UG)6 RNA with increasing concentrations of GST-TDP (101–261), and this could be interpreted as a change in RNA conformation due to TDP-43 binding (Figure 4A). Compared with this situation, the subtracted CD spectra of CLIP34nt displayed subtle changes at 265 nm, whereas the subtracted spectra of CLIP6 RNA showed that the negative peak at 210 nm (in unbound state) shifted to 200 nm on binding with protein, but the peak at 268 nm remained unchanged. These subtle changes may be because of the presence of comparatively weak interaction of CLIP34nt and CLIP6 with GST-TDP (101–261) with respect to (UG)6 RNA (Figure 4B and C). Finally, in the case of the CLIP34nt_UG6 RNA sample, it was observed that the intensity at 255 nm decreased with increasing GST-TDP (101–261) concentrations like it was observed with (UG)6 RNA (Figure 4D). The CD titration experiment was also performed with GST-TDPmut2 using CLIP34nt_UG6 RNA, and this mutant displayed the same binding affinity as GST-TDP (101–261) to all the target RNA oligos. The substracted spectra displayed the same pattern like the one observed for GST-TDP (101–261), with just some subtle difference at 15 μM protein concentration (Figure 4E).Figure 4.

Bottom Line: Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself.In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop.In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), 34012 Trieste, Italy.

ABSTRACT
One of the most important functional features of nuclear factor TDP-43 is its ability to bind UG-repeats with high efficiency. Several cross-linking and immunoprecipitation (CLIP) and RNA immunoprecipitation-sequencing (RIP-seq) analyses have indicated that TDP-43 in vivo can also specifically bind loosely conserved UG/GU-rich repeats interspersed by other nucleotides. These sequences are predominantly localized within long introns and in the 3'UTR of various genes. Most importantly, some of these sequences have been found to exist in the 3'UTR region of TDP-43 itself. In the TDP-43 3'UTR context, the presence of these UG-like sequences is essential for TDP-43 to autoregulate its own levels through a negative feedback loop. In this work, we have compared the binding of TDP-43 with these types of sequences as opposed to perfect UG-stretches. We show that the binding affinity to the UG-like sequences has a dissociation constant (Kd) of ∼110 nM compared with a Kd of 8 nM for straight UGs, and have mapped the region of contact between protein and RNA. In addition, our results indicate that the local concentration of UG dinucleotides in the CLIP sequences is one of the major factors influencing the interaction of these RNA sequences with TDP-43.

Show MeSH
Related in: MedlinePlus