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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

Extracted ion Chromatograms of acetylated His-TDP (101–261). The extracted ion chromatograms for the acetylated chymotryptic peptide 132MVQKKDLKTGHSKGF147 (Chym132–147) were generated for samples in the absence and presence of RNA. Importantly, the light acetylation was used to label TDP-43 in the absence of RNA (red traces, Peak ‘L’), while the heavy reagent was used to label TDP-43 in the presence of RNA (blue traces, Peak ‘H’). For each binary comparison, the samples were mixed before chymotryptic digestion and analyzed in the same LC-MS/MS run. The production of the acetylated Chym132–147 peptides from no RNA controls are shown compared with TDP-43 + (UG)6 (A), TDP-43 + CLIP34nt (B) and TDP-43 + CLIP6 (C). The x-axis shows the intensity of the eluting peptides and, in all cases, the presence of RNA inhibited the acetylation of Lysine145 (underlined) as indicated by the greater intensity of the blue traces relative to the red traces. y-axis shows the elution time of these peptides.
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gkt189-F3: Extracted ion Chromatograms of acetylated His-TDP (101–261). The extracted ion chromatograms for the acetylated chymotryptic peptide 132MVQKKDLKTGHSKGF147 (Chym132–147) were generated for samples in the absence and presence of RNA. Importantly, the light acetylation was used to label TDP-43 in the absence of RNA (red traces, Peak ‘L’), while the heavy reagent was used to label TDP-43 in the presence of RNA (blue traces, Peak ‘H’). For each binary comparison, the samples were mixed before chymotryptic digestion and analyzed in the same LC-MS/MS run. The production of the acetylated Chym132–147 peptides from no RNA controls are shown compared with TDP-43 + (UG)6 (A), TDP-43 + CLIP34nt (B) and TDP-43 + CLIP6 (C). The x-axis shows the intensity of the eluting peptides and, in all cases, the presence of RNA inhibited the acetylation of Lysine145 (underlined) as indicated by the greater intensity of the blue traces relative to the red traces. y-axis shows the elution time of these peptides.

Mentions: In our experiments, we found that TDP-43 lysine labelling with acetic anhydride to be robust and gentle and did not interfere with RNA gel shifting at concentration up to 5 mM (data not shown). In addition, binary comparisons can be performed via mass spectrometry using different stable isotope versions of acetic anhydride. The schematic representation of the work flow is shown in Supplementary Figure S2. Briefly, free His-TDP (101–261) was treated with 5 mM acetic anhydride and His-TDP (101–261) in the presence of RNA was treated with equimolar concentration of hexadeuteroacetic anhydride (heavy). The two reactions were mixed, separated by SDS-PAGE and subjected to in-gel digestion with chymotrypsin, which resulted in a mixture of acetylated and heavy acetyl derivative of the same peptides. These peptides were then separated and analyzed by LC-MS/MS. Acetylated and heavy acetylated peptides were identified based on their mass shifts of 42 and 45 Da per lysine residue, respectively. In addition, unacetylated lysine residues were also found indicating that these lysines are buried in the structure and have poor solvent accessibility in the test conditions. To maintain the homogeneity in the text, we used the amino acid numbering as per the full-length wild-type TDP-43. We were particularly interested in the Chym132-147 peptide (132MVQVKKDLKTGHSKGF147), which contains part of the RRM1 and includes the Phe147 residue required for RNA binding. For our purposes, Chym132-147 was of considerable importance as it originates by the chymotryptic digestion at Phe147, dissecting the active site residues of RRM1, and hence can provide information about RNA recognition and binding. In the case of free His-TDP (101–261), we observed that all the four lysine residues were labelled uniformly with acetic anhydride, indicating that in the absence of nucleic acid, these lysines are solvent accessible. However, in case of His-TDP–(UG)6, His-TDP–CLIP34nt and His-TDP–CLIP6 complex, we observed partial labelling of K145 (underlined in Figure 3) with d3-acetic anhydride (Figure 3). Comparative analysis of the light and heavy form of this peptide clearly showed that partial labelling of K145 was greatly enhanced by the presence of RNA as compared with free His-TDP (Figure 3). These results demonstrate that the binding to these RNAs protects K145 residues and makes it less accessible for acetic anhydride labelling. All the results obtained using this technique concern RRM1. The role of RRM2 in binding to RNA is still unknown although it has been previously claimed that low affinity RNA or DNA binding may occur through the RRM2 (12). This strategy does not provide any appropriate answer about the role of RRM2 domain in RNA binding because the nearest lysine residue is four residues away and lies on a different chymotryptic peptide.Figure 3.


Characterizing TDP-43 interaction with its RNA targets.

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

Extracted ion Chromatograms of acetylated His-TDP (101–261). The extracted ion chromatograms for the acetylated chymotryptic peptide 132MVQKKDLKTGHSKGF147 (Chym132–147) were generated for samples in the absence and presence of RNA. Importantly, the light acetylation was used to label TDP-43 in the absence of RNA (red traces, Peak ‘L’), while the heavy reagent was used to label TDP-43 in the presence of RNA (blue traces, Peak ‘H’). For each binary comparison, the samples were mixed before chymotryptic digestion and analyzed in the same LC-MS/MS run. The production of the acetylated Chym132–147 peptides from no RNA controls are shown compared with TDP-43 + (UG)6 (A), TDP-43 + CLIP34nt (B) and TDP-43 + CLIP6 (C). The x-axis shows the intensity of the eluting peptides and, in all cases, the presence of RNA inhibited the acetylation of Lysine145 (underlined) as indicated by the greater intensity of the blue traces relative to the red traces. y-axis shows the elution time of these peptides.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt189-F3: Extracted ion Chromatograms of acetylated His-TDP (101–261). The extracted ion chromatograms for the acetylated chymotryptic peptide 132MVQKKDLKTGHSKGF147 (Chym132–147) were generated for samples in the absence and presence of RNA. Importantly, the light acetylation was used to label TDP-43 in the absence of RNA (red traces, Peak ‘L’), while the heavy reagent was used to label TDP-43 in the presence of RNA (blue traces, Peak ‘H’). For each binary comparison, the samples were mixed before chymotryptic digestion and analyzed in the same LC-MS/MS run. The production of the acetylated Chym132–147 peptides from no RNA controls are shown compared with TDP-43 + (UG)6 (A), TDP-43 + CLIP34nt (B) and TDP-43 + CLIP6 (C). The x-axis shows the intensity of the eluting peptides and, in all cases, the presence of RNA inhibited the acetylation of Lysine145 (underlined) as indicated by the greater intensity of the blue traces relative to the red traces. y-axis shows the elution time of these peptides.
Mentions: In our experiments, we found that TDP-43 lysine labelling with acetic anhydride to be robust and gentle and did not interfere with RNA gel shifting at concentration up to 5 mM (data not shown). In addition, binary comparisons can be performed via mass spectrometry using different stable isotope versions of acetic anhydride. The schematic representation of the work flow is shown in Supplementary Figure S2. Briefly, free His-TDP (101–261) was treated with 5 mM acetic anhydride and His-TDP (101–261) in the presence of RNA was treated with equimolar concentration of hexadeuteroacetic anhydride (heavy). The two reactions were mixed, separated by SDS-PAGE and subjected to in-gel digestion with chymotrypsin, which resulted in a mixture of acetylated and heavy acetyl derivative of the same peptides. These peptides were then separated and analyzed by LC-MS/MS. Acetylated and heavy acetylated peptides were identified based on their mass shifts of 42 and 45 Da per lysine residue, respectively. In addition, unacetylated lysine residues were also found indicating that these lysines are buried in the structure and have poor solvent accessibility in the test conditions. To maintain the homogeneity in the text, we used the amino acid numbering as per the full-length wild-type TDP-43. We were particularly interested in the Chym132-147 peptide (132MVQVKKDLKTGHSKGF147), which contains part of the RRM1 and includes the Phe147 residue required for RNA binding. For our purposes, Chym132-147 was of considerable importance as it originates by the chymotryptic digestion at Phe147, dissecting the active site residues of RRM1, and hence can provide information about RNA recognition and binding. In the case of free His-TDP (101–261), we observed that all the four lysine residues were labelled uniformly with acetic anhydride, indicating that in the absence of nucleic acid, these lysines are solvent accessible. However, in case of His-TDP–(UG)6, His-TDP–CLIP34nt and His-TDP–CLIP6 complex, we observed partial labelling of K145 (underlined in Figure 3) with d3-acetic anhydride (Figure 3). Comparative analysis of the light and heavy form of this peptide clearly showed that partial labelling of K145 was greatly enhanced by the presence of RNA as compared with free His-TDP (Figure 3). These results demonstrate that the binding to these RNAs protects K145 residues and makes it less accessible for acetic anhydride labelling. All the results obtained using this technique concern RRM1. The role of RRM2 in binding to RNA is still unknown although it has been previously claimed that low affinity RNA or DNA binding may occur through the RRM2 (12). This strategy does not provide any appropriate answer about the role of RRM2 domain in RNA binding because the nearest lysine residue is four residues away and lies on a different chymotryptic peptide.Figure 3.

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