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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

Circular dichroism analysis of GST-TDP (101–261) in the presence of RNA: (A) lane 1 of coomassie-stained EMSA gel shows the mobility of free GST-TDP (101–261) (arrow 1). In lane 2, a faster-moving band appeared in the presence of (UG)6 RNA (arrow 2), indicating the formation of a stable protein–RNA complex. Mobility of GST-TDP (101–261) did not change in the presence of CLIP1 RNA (negative control, lane3). Lane (4–5) shows that the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear indicating the formation of weak complex. (B) All the samples from A were subjected to circular dichroism, and no significant changes were observed. (C) Appearance of a faster-moving band showing the stable complex between GST-TDP (101–261) and CLIP34nt_UG. (D) Far-UV CD spectra of GST-TDP (101–261) in the presence of CLIP34nt_UG.
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gkt189-F5: Circular dichroism analysis of GST-TDP (101–261) in the presence of RNA: (A) lane 1 of coomassie-stained EMSA gel shows the mobility of free GST-TDP (101–261) (arrow 1). In lane 2, a faster-moving band appeared in the presence of (UG)6 RNA (arrow 2), indicating the formation of a stable protein–RNA complex. Mobility of GST-TDP (101–261) did not change in the presence of CLIP1 RNA (negative control, lane3). Lane (4–5) shows that the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear indicating the formation of weak complex. (B) All the samples from A were subjected to circular dichroism, and no significant changes were observed. (C) Appearance of a faster-moving band showing the stable complex between GST-TDP (101–261) and CLIP34nt_UG. (D) Far-UV CD spectra of GST-TDP (101–261) in the presence of CLIP34nt_UG.

Mentions: Regarding the effects of RNA binding on protein conformation, it has been shown that during the formation of most protein–RNA complexes, the protein, the RNA and sometimes both can undergo conformational changes such as with the TAR–TAT complex (34) and U1A–RNA complex (24,34). However, no such study has been carried out with respect to the TDP-43–(UG)n complex or TDP-43–CLIP complex. Therefore, in parallel to these analyses, we also analyzed the conformational changes in the GST-TDP (101–261) protein itself following RNA interaction using circular dichroism. The far-UV CD spectra of GST-TDP (101–261) alone showed two negative peaks at 208 and 222 nm, characteristic of α-helical structure (Figure 5B). It is evident from the CD analysis performed at fixed protein and RNA concentrations that the resultant subtracted CD spectra of GST-TDP (101–261) did not show any significant conformational changes in the presence of the various RNA oligonucleotides (Figure 5B–D). In fact, considering that both protein and RNA contribute at 208 nm, even the small changes observed around 208 nm in the subtracted CD spectra of GST-TDP cannot be attributed solely to a change in protein conformation alone. Nevertheless, subtle localized changes in GST-TDP (101–261) conformation cannot be ruled out and more advanced studies (i.e. NMR or radiographic crystallography) might be required to investigate this possibility. To confirm that a significant amount of GST-TDP (101–261) was bound to RNA while recording the CD spectrum, we performed the native EMSA gels using the same samples prepared during CD analysis. Figure 5A shows the mobility of free GST-TDP (101–261) under coomassie-stained native EMSA gel (lane 2, marked as ‘free’). In Figure 5A, lane 3, a faster-moving band appeared in the presence of (UG)6 RNA (marked as ‘bound’), indicating the formation of a stable protein–RNA complex. As expected, mobility of TDP-43 did not change in the presence of CLIP1 RNA (non-binding control, lane 4), and the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear, which is often indicative of the formation of a weak complex (Figure 5A, lane 5–6). The appearance of a well-defined faster-moving band was also observed using this technique for GST-TDP-CLIP34nt_UG (Figure 5C, lane 2–3).Figure 5.


Characterizing TDP-43 interaction with its RNA targets.

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

Circular dichroism analysis of GST-TDP (101–261) in the presence of RNA: (A) lane 1 of coomassie-stained EMSA gel shows the mobility of free GST-TDP (101–261) (arrow 1). In lane 2, a faster-moving band appeared in the presence of (UG)6 RNA (arrow 2), indicating the formation of a stable protein–RNA complex. Mobility of GST-TDP (101–261) did not change in the presence of CLIP1 RNA (negative control, lane3). Lane (4–5) shows that the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear indicating the formation of weak complex. (B) All the samples from A were subjected to circular dichroism, and no significant changes were observed. (C) Appearance of a faster-moving band showing the stable complex between GST-TDP (101–261) and CLIP34nt_UG. (D) Far-UV CD spectra of GST-TDP (101–261) in the presence of CLIP34nt_UG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt189-F5: Circular dichroism analysis of GST-TDP (101–261) in the presence of RNA: (A) lane 1 of coomassie-stained EMSA gel shows the mobility of free GST-TDP (101–261) (arrow 1). In lane 2, a faster-moving band appeared in the presence of (UG)6 RNA (arrow 2), indicating the formation of a stable protein–RNA complex. Mobility of GST-TDP (101–261) did not change in the presence of CLIP1 RNA (negative control, lane3). Lane (4–5) shows that the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear indicating the formation of weak complex. (B) All the samples from A were subjected to circular dichroism, and no significant changes were observed. (C) Appearance of a faster-moving band showing the stable complex between GST-TDP (101–261) and CLIP34nt_UG. (D) Far-UV CD spectra of GST-TDP (101–261) in the presence of CLIP34nt_UG.
Mentions: Regarding the effects of RNA binding on protein conformation, it has been shown that during the formation of most protein–RNA complexes, the protein, the RNA and sometimes both can undergo conformational changes such as with the TAR–TAT complex (34) and U1A–RNA complex (24,34). However, no such study has been carried out with respect to the TDP-43–(UG)n complex or TDP-43–CLIP complex. Therefore, in parallel to these analyses, we also analyzed the conformational changes in the GST-TDP (101–261) protein itself following RNA interaction using circular dichroism. The far-UV CD spectra of GST-TDP (101–261) alone showed two negative peaks at 208 and 222 nm, characteristic of α-helical structure (Figure 5B). It is evident from the CD analysis performed at fixed protein and RNA concentrations that the resultant subtracted CD spectra of GST-TDP (101–261) did not show any significant conformational changes in the presence of the various RNA oligonucleotides (Figure 5B–D). In fact, considering that both protein and RNA contribute at 208 nm, even the small changes observed around 208 nm in the subtracted CD spectra of GST-TDP cannot be attributed solely to a change in protein conformation alone. Nevertheless, subtle localized changes in GST-TDP (101–261) conformation cannot be ruled out and more advanced studies (i.e. NMR or radiographic crystallography) might be required to investigate this possibility. To confirm that a significant amount of GST-TDP (101–261) was bound to RNA while recording the CD spectrum, we performed the native EMSA gels using the same samples prepared during CD analysis. Figure 5A shows the mobility of free GST-TDP (101–261) under coomassie-stained native EMSA gel (lane 2, marked as ‘free’). In Figure 5A, lane 3, a faster-moving band appeared in the presence of (UG)6 RNA (marked as ‘bound’), indicating the formation of a stable protein–RNA complex. As expected, mobility of TDP-43 did not change in the presence of CLIP1 RNA (non-binding control, lane 4), and the addition of CLIP34nt and CLIP6 resulted in the appearance of a smear, which is often indicative of the formation of a weak complex (Figure 5A, lane 5–6). The appearance of a well-defined faster-moving band was also observed using this technique for GST-TDP-CLIP34nt_UG (Figure 5C, lane 2–3).Figure 5.

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