Difference between revisions of "Part:BBa K3407004"
Line 5: Line 5:
  
 
Fox-1 stands for Feminizing Locus on X, a protein acting as a numerator element to count the number of X chromosomes relative to ploidy. In these worms, Fox-1 is thought to determine sex by post-transcriptional repression of Xol-1. In mammalian genes, it strongly influences the splicing of alternative exons by binding to “UGCAUG” RNA sequence <html><a href="#1">[1]</a></html>. Orthologs are present in a wide range of multicellular eukarya species like zebrafish <html><a href="#2">[2]</a></html>, mice <html><a href="#3">[3]</a></html>, rats <html><a href="#4">[4]</a></html>, bulls <html><a href="#5">[5]</a></html>, frogs <html><a href="#6">[6]</a></html>, chicken <html><a href="#7">[7]</a></html>, polar bear <html><a href="#8">[8]</a></html>, cats <html><a href="#9">[9]</a></html>, platypuses <html><a href="#10">[10]</a></html>, sunfishes <html><a href="#11">[11]</a></html>, tasmanian devils <html><a href="#12">[12]</a></html> and humans <html><a href="#13">[13]</a></html>.
 
Fox-1 stands for Feminizing Locus on X, a protein acting as a numerator element to count the number of X chromosomes relative to ploidy. In these worms, Fox-1 is thought to determine sex by post-transcriptional repression of Xol-1. In mammalian genes, it strongly influences the splicing of alternative exons by binding to “UGCAUG” RNA sequence <html><a href="#1">[1]</a></html>. Orthologs are present in a wide range of multicellular eukarya species like zebrafish <html><a href="#2">[2]</a></html>, mice <html><a href="#3">[3]</a></html>, rats <html><a href="#4">[4]</a></html>, bulls <html><a href="#5">[5]</a></html>, frogs <html><a href="#6">[6]</a></html>, chicken <html><a href="#7">[7]</a></html>, polar bear <html><a href="#8">[8]</a></html>, cats <html><a href="#9">[9]</a></html>, platypuses <html><a href="#10">[10]</a></html>, sunfishes <html><a href="#11">[11]</a></html>, tasmanian devils <html><a href="#12">[12]</a></html> and humans <html><a href="#13">[13]</a></html>.
−
 
  
 
<div><ul>  
 
<div><ul>  
 
<center>
 
<center>
−
  <li style="display: inline-block;"> [[File:T--TUDelft--Fox_Xray.png|thumb|none|250px|<b>Figure 1:</b> Crystal structure of Fox-1 RBD bound to its UGCAUGU target sequence. The structure has been resolved <html><a href="#1">[1]</a></html> available in PDB with 2ERR accession number <html><a href="#15">[15]</a></html>.]] </li>
+
  <li style="display: inline-block;"> [[File:T--TUDelft--Fox_Xray.png|thumb|none|250px|<b>Figure 1:</b> Crystal structure of Fox-1 RBD bound to its UGCAUGU target sequence. The structure has been resolved <html><a href="#1">[1]</a></html> available in PDB with 2ERR accession number <html><a href="#15">[15]</a></html>.]] </li>
−
</center>
+
</center>
 
     </ul></div>
 
     </ul></div>
  
Line 31: Line 30:
 
     </ul></div>
 
     </ul></div>
  
−
For each short hairpin, a pair of complementary primers were annealed to form a DNA template for transcription (Figure 2B). The DNA template was designed to possess a T7 promoter followed by a 27nt inverted repeat sequence taken from the eGFP gene (nt 78 to 105), and linked together by the 9nt sequence containing the target of Fox-1 RBD. On the 3’ termini, two GG were added to produce the desired overhang (<html><a href="https://parts.igem.org/Part:BBa_K3407002" target="_blank"><b>BBa_K3407022</b></a></html>). This leads to a successful transcription without the need to use a terminator (Figure 2A).
+
Additional protein bands corresponding to molecular weights of 13.6 kDa (Fox-1 RBD) and 13.4 kDa (Fox-1 RBD*, mutated) were observed after induction (Figure 2A), indicating that both proteins were overexpressed. Moreover, a clear band is obtained in the elution samples (E) (Figure 2B), demonstrating that both Fox-1 RBD and RBD* were successfully purified.  
−
 
+
−
<div><ul>
+
−
<center>
+
−
  <li style="display: inline-block;"> [[File:T--TUDelft--shRNA_Results.jpeg|thumb|none|800px|<b>Figure 2:</b> Schematic representation of the shRNA in vitro production using T7 RiboMAX kit from Promega. Primer pairs are annealed in the T4 ligase buffer by heating at 95ºC for 5 minutes and left cooling at room temperature for 30 minutes. Once template is annealed, T7 RNA polymerase is added to transcribe the DNA template at 42ºC for 2 hours. shRNAs self assemble as soon as they are produced. (B) DNA template design for in vitro shRNA production. T7 promoter followed by eGFP target sequences with inverted repeats are linked by the loop region, and finally ends with a GG overhang.]] </li>
+
−
</center>
+
−
    </ul></div>
+
  
 
===References===
 
===References===

Revision as of 16:00, 26 October 2020

Fox-1 RBD: a protein domain binding strongly and specifically to RNA sequence UGCAUGU.

Usage and Biology

In many projects involving RNA approaches, the availability of a library of proteins able to bind strongly and specifically to an RNA target sequence represents a valuable and versatile set of BioBricks. Here we describe Fox-1 RBD, a protein domain able to recognise and bind a specific RNA sequence.

Fox-1 stands for Feminizing Locus on X, a protein acting as a numerator element to count the number of X chromosomes relative to ploidy. In these worms, Fox-1 is thought to determine sex by post-transcriptional repression of Xol-1. In mammalian genes, it strongly influences the splicing of alternative exons by binding to “UGCAUG” RNA sequence [1]. Orthologs are present in a wide range of multicellular eukarya species like zebrafish [2], mice [3], rats [4], bulls [5], frogs [6], chicken [7], polar bear [8], cats [9], platypuses [10], sunfishes [11], tasmanian devils [12] and humans [13].

  • Figure 1: Crystal structure of Fox-1 RBD bound to its UGCAUGU target sequence. The structure has been resolved [1] available in PDB with 2ERR accession number [15].

This protein has a domain of interaction with RNA called RNA Binding Domain (RBD), also commonly referred as RNA Recognition Motif (RRM) and Ribonucleoprotein (RNP) domain, referring to the domain formed by the residues 109 to 208 of Fox-1 (13.6 kDa when his-tagged). Its RBD has shown a high affinity (Kd = 0.49 nM at 150mM NaCl) to “UGCAUGU” RNA sequence where residues F126 and F160 play a crucial role in its binding capacity [1][14]. Interestingly, it has also shown a high specificity to the mentioned sequence where single mutations can reduce from 4,8 to more than 1.500 fold the affinity with its substrate [1], becoming a powerful BioBrick where a highly specific and affine interaction is needed between a protein and RNA. As shown in its crystal structure, C-terminal of the RBD is distant from the binding site and can accept a His-tag without showing a negative effect on the binding capacity [15], probably providing an adequate locus to fuse other domains or proteins. A mutated version of the protein domain has been expressed and tested as a control, where mutations F160A & F126A deplete its binding capacity [1] (BBa_K3407004).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental results

Fox-1 RBD and RBD* (mutated) overexpression and purification

In order to check the overexpression of Fox-1 RBD and its mutated version (BBa_K3407004), we incubated the E. coli BL21 (DE3) (Negative control) and E. coli BL21 (DE3) transformed either with the pBbB7a_Fox-1_RBD (BBa_K3407020) or pBbB7a_Fox-1_RBD* (BBa_K3407024) at 37ºC until the OD600 reached ~ 0.6. We induced the cultures with a final IPTG concentration of 1 mM and incubated them 4h at 37ºC and overnight at 30ºC. The total protein content of the cells was analysed by SDS-PAGE electrophoresis (Figure 2A). As the recombinantly expressed Fox-1 RBD and RBD* (mutated) contain a His-tag at their N-terminal part, we purified them using affinity chromatography with the HisLink protein purification system (Promega). Samples obtained from the different purification steps were analysed by SDS-PAGE electrophoresis (Figure 2B).

  • Figure 2: Purification of Fox-1 RBD and mutated Fox-1 RBD*. (A) SDS-PAGE gel of the overexpression samples before and after induction with IPTG. (B) SDS-PAGE of samples from the different purification steps. E. coli BL21 (DE3) is the negative control, E. coli BL21 (DE3) Fox-1 RBD contains the part BBa_K3407020 and E. coli BL21 (DE3) Fox-1 RBD* contains the part BBa_K3407024. MW (Molecular weight marker, #1610363 Bio-Rad), PI (pre-induction), 4h (4 hours after induction), ON (overnight), FT (flow-through), W1 and W2 (Washing), E (Elution). All the samples used corresponded to the same OD600.

Additional protein bands corresponding to molecular weights of 13.6 kDa (Fox-1 RBD) and 13.4 kDa (Fox-1 RBD*, mutated) were observed after induction (Figure 2A), indicating that both proteins were overexpressed. Moreover, a clear band is obtained in the elution samples (E) (Figure 2B), demonstrating that both Fox-1 RBD and RBD* were successfully purified.

References

Ordered List

  1. Auweter, S., Fasan, R., Reymond, L., Underwood, J., Black, D., Pitsch, S. and Allain, F., 2020. Molecular Basis Of RNA Recognition By The Human Alternative Splicing Factor Fox-1.
  2. UniProtKB - Q642J5 (RFOX1_DANRE)
  3. UniProtKB - Q9JJ43 (RFOX1_MOUSE)
  4. UniProtKB - A1A5R1 (RFOX2_RAT)
  5. UniProtKB - A6QPR6 (RFOX2_BOVIN)
  6. UniProtKB - Q66JB7 (RFOX2_XENTR)
  7. UniProtKB - F1NCA4 (F1NCA4_CHICK)
  8. UniProtKB - A0A384DA29 (A0A384DA29_URSMA)
  9. UniProtKB - A0A337SR52 (A0A337SR52_FELCA)
  10. UniProtKB - F6SR19 (F6SR19_ORNAN)
  11. UniProtKB - A0A3Q4B4Y5 (A0A3Q4B4Y5_MOLML)
  12. UniProtKB - G3VXH1 (G3VXH1_SARHA)
  13. UniProtKB - Q9NWB1 (RFOX1_HUMAN)
  14. Jin, Y., 2020. A Vertebrate RNA-Binding Protein Fox-1 Regulates Tissue-Specific Splicing Via The Pentanucleotide GCAUG.
  15. NMR Structure of the RNA Binding Domain of Human Fox-1 in Complex with UGCAUGU