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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.

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Related in: MedlinePlus

Quantitative EMSA analysis of various TDP–RNA complexes. Panel (A–C) shows the gel profiles and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDP (101–261). Panel (D–F) show the gel profile and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDPmut2 (101–261 F229L and F231L). The concentration (nM) of each probe used to determine the Kd is mentioned on the top of each gel. Each experiment was repeated at least three times to plot the binding curves.
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gkt189-F2: Quantitative EMSA analysis of various TDP–RNA complexes. Panel (A–C) shows the gel profiles and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDP (101–261). Panel (D–F) show the gel profile and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDPmut2 (101–261 F229L and F231L). The concentration (nM) of each probe used to determine the Kd is mentioned on the top of each gel. Each experiment was repeated at least three times to plot the binding curves.

Mentions: Subsequently, the Kd values for CLIP34nt, CLIP6 and CLIP34nt_UG6 were calculated using GST-TDP (101–261) using quantitative EMSA (27) (Figure 2, Table 1). For this experiment, we also used a GST-TDPmut2 (101–261), which was previously shown to bind to UG-repeated sequences in EMSA analysis (11). This mutant was chosen to gain additional quantitative insight in the role played by RRM2 Phenylalanines 229 and 231 in the binding of TDP-43 to CLIPs and UG6. Using this mutant, we observed that all Kd values for all the CLIP sequences were comparable with those observed for GST-TDP (101–261). This result suggested that, unlike Phenylalanines 147 and 149 in RRM1 (11), the corresponding residues of RRM2 do not play a major role in the recognition and binding of TDP-43 with any of these sequences (Table 1).Figure 2.


Characterizing TDP-43 interaction with its RNA targets.

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

Quantitative EMSA analysis of various TDP–RNA complexes. Panel (A–C) shows the gel profiles and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDP (101–261). Panel (D–F) show the gel profile and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDPmut2 (101–261 F229L and F231L). The concentration (nM) of each probe used to determine the Kd is mentioned on the top of each gel. Each experiment was repeated at least three times to plot the binding curves.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt189-F2: Quantitative EMSA analysis of various TDP–RNA complexes. Panel (A–C) shows the gel profiles and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDP (101–261). Panel (D–F) show the gel profile and binding curves plotted using quantitative EMSA analysis for CLIP34nt_UG6, CLIP34nt and CLIP6 with GST-TDPmut2 (101–261 F229L and F231L). The concentration (nM) of each probe used to determine the Kd is mentioned on the top of each gel. Each experiment was repeated at least three times to plot the binding curves.
Mentions: Subsequently, the Kd values for CLIP34nt, CLIP6 and CLIP34nt_UG6 were calculated using GST-TDP (101–261) using quantitative EMSA (27) (Figure 2, Table 1). For this experiment, we also used a GST-TDPmut2 (101–261), which was previously shown to bind to UG-repeated sequences in EMSA analysis (11). This mutant was chosen to gain additional quantitative insight in the role played by RRM2 Phenylalanines 229 and 231 in the binding of TDP-43 to CLIPs and UG6. Using this mutant, we observed that all Kd values for all the CLIP sequences were comparable with those observed for GST-TDP (101–261). This result suggested that, unlike Phenylalanines 147 and 149 in RRM1 (11), the corresponding residues of RRM2 do not play a major role in the recognition and binding of TDP-43 with any of these sequences (Table 1).Figure 2.

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