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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

Proposed binding mechanism of TDP-43 (via RRM1) and multi-site RNA targets containing UG-repeats. In this model, long UG-repeats maintain a high local concentration of UGs, which in turn increases the probability of binding other shorter or single UGs stretches that prevent the protein to diffuse away before rebinding can occur.
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gkt189-F6: Proposed binding mechanism of TDP-43 (via RRM1) and multi-site RNA targets containing UG-repeats. In this model, long UG-repeats maintain a high local concentration of UGs, which in turn increases the probability of binding other shorter or single UGs stretches that prevent the protein to diffuse away before rebinding can occur.

Mentions: On the basis of the present results and on previous data, which suggested that Phe147 and Phe 149 make aromatic stacking interaction with uracil (U) and guanine (G), respectively (27), we are proposing a binding model for the TDP–RNA complex (Figure 6). According to this model, as TDP-43 gets near to an RNA, its RRM1 domain binds and falls off from UG nucleotides present in the sequence (Figure 6A). In keeping with this, it should be noted the differential Kd values for various (UG)n-repeats suggests that off-rate (Kd) from one or two UGs is fast and hence could not be determined (Tables 1 and 2). Longer repeats would be important in maintaining higher local concentration of UGs, thus providing ample opportunity for TDP-43 to bind another UG once dissociated from the previous one. As a result, the dissociation rate (Kd) decreases with the increasing length of (UG)n-repeats (Figure 6B). Finally, it should also be noted that besides the key interaction of TDP-43 RRM1 with a UG sequence, several other factors may influence the binding of this protein in vivo. For example, RNA secondary and tertiary structures may also provide another controlling factor during the formation of TDP-43–RNA complex and is well supported by our CD analysis. Furthermore, it has been shown, for example, that TDP-43 does not interact with UG-repeats if they are extensively involved in base pairing (40) and may explain the ambiguity behind the unpredictable nature of TDP-43 binding to apparently long UG-repeats.Figure 6.


Characterizing TDP-43 interaction with its RNA targets.

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

Proposed binding mechanism of TDP-43 (via RRM1) and multi-site RNA targets containing UG-repeats. In this model, long UG-repeats maintain a high local concentration of UGs, which in turn increases the probability of binding other shorter or single UGs stretches that prevent the protein to diffuse away before rebinding can occur.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt189-F6: Proposed binding mechanism of TDP-43 (via RRM1) and multi-site RNA targets containing UG-repeats. In this model, long UG-repeats maintain a high local concentration of UGs, which in turn increases the probability of binding other shorter or single UGs stretches that prevent the protein to diffuse away before rebinding can occur.
Mentions: On the basis of the present results and on previous data, which suggested that Phe147 and Phe 149 make aromatic stacking interaction with uracil (U) and guanine (G), respectively (27), we are proposing a binding model for the TDP–RNA complex (Figure 6). According to this model, as TDP-43 gets near to an RNA, its RRM1 domain binds and falls off from UG nucleotides present in the sequence (Figure 6A). In keeping with this, it should be noted the differential Kd values for various (UG)n-repeats suggests that off-rate (Kd) from one or two UGs is fast and hence could not be determined (Tables 1 and 2). Longer repeats would be important in maintaining higher local concentration of UGs, thus providing ample opportunity for TDP-43 to bind another UG once dissociated from the previous one. As a result, the dissociation rate (Kd) decreases with the increasing length of (UG)n-repeats (Figure 6B). Finally, it should also be noted that besides the key interaction of TDP-43 RRM1 with a UG sequence, several other factors may influence the binding of this protein in vivo. For example, RNA secondary and tertiary structures may also provide another controlling factor during the formation of TDP-43–RNA complex and is well supported by our CD analysis. Furthermore, it has been shown, for example, that TDP-43 does not interact with UG-repeats if they are extensively involved in base pairing (40) and may explain the ambiguity behind the unpredictable nature of TDP-43 binding to apparently long UG-repeats.Figure 6.

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