Connector element

• C end of Torus and N end of RRM linker

• Loop (residues 116-133) with a6 at its end  

Figure 1. Structure of connector loop in Cwc2, conserved positively charged residues highlighted

• Involved in RNA recognition? ~ Conserved residues (compare to RNA motifs/complementarity to RNA)

The possibility of the connector loop being involved in RNA recognition was explored by carrying out a BLAST on its sequence. 

Figure 2. BLAST results for connector loop

We can see that we are not getting results with known proteins with RNA-binding motifs. Most of the results are hypothetical proteins characterised from open reading frames. Perhaps further research on possible domains of these proteins might lend insight into the possibility of the connector loop interacting RNA. Evidence for this was obtained from mutagenesis experiments on the connector loop. (Lu et al, 2012)

However, the loop might still function to guide RNA to the depression (pocket) and this is explored below.   

• Contains positively charged patch, interconnected to patches on RRM and ZnF 

Figure 3. Electrostatic surface potential of Cwc2

The blue patches show the positively charged regions on the connector loop, around the ZnF and the RRM (from top to bottom). The patches are linked by positively charged residues. The patches line depression and could be said to hold RNA secondary structure. This charge distribution on the surface could be vital to guiding the negatively charged RNA in the right conformation to the binding sites, thereby making binding more efficient.

• Connector form rear rim and is on top of depression ~ guide RNA to pocket? 

We see that there are many potential RNA binding sites predicted and are present in the connector loop as well. However this is only a rough gauge and further experimentation involving isolated connector loop and RNA binding assays needs to be carried out. But we can say with some confidence the connector loop is involved in guiding the RNA to the binding pocket, thereby increasing efficiency of Cwc2.

• Crosslinks with U6 snRNA ~ all contacts: ZnF (C87), the connector element (Y120–R121), the RRM (K152, C181 from RNP1 and Y138 from RNP2) and in the Torus domain (F47)  

Figure 4. Binding site to U6 snRNA characterised by crosslinking highlighted residues

We can see from above that the shown top view face of the molecule is implicated in binding and assisting in splicing to form mature U6 snRNA. Other literature indicates the importance of Cwc2 in this function. Furthermore it is seen that U6 snRNA is not the only snRNA involved in regulation by Cwc2. 

Figure 5. Gel run of glucose induced Cwc2 gene-repressed yeast cell extract (Mcgrail. et al, 2009

Upon switching from inducing to repressing Cwc2 expression under Gal1 promoter, we see that the levels of U1, U4, U5 and U6 snRNA levels drop indicating possible interactions in these snRNAs with Cwc2. 

Figure 6. Gel shift assay using non-denaturing PAGE with snRNA and Cwc2

We see that there is specific binding and slower movement of complex by Cwc2 binding to U1, U2, U4 and U5 snRNA. The results shown in Figure 5 are supported by Figure 6 results. Furthermore, Cwc2 was shown to crosslink with U6 snRNA using immunoblotting with anti-TAP antibody against TAP-tagged (tandem affinity purification) Cwc2. Cwc2-TAP crosslinked to U6 snRNA in the first and second steps of spliceosome formation in yeast. Thus we can postulate that Cwc2 functions in binding U6 to spliceosome upon U4 dissociating and directing its interactions with pre-mRNA. (McGrail et al, 2009) It was also found that the connector and ZnF bind U6 than U4 snRNA with greater affinity. The whole complex serves to carry out pre-mRNA splicing activity and Cwc2 plays an important role in mediating it. This is seen by the point mutation Y120A removing splicing activity. We can thus tell that the hydrophobic interactions set up by the tyrosine-120 residue is key to positioning spliceosome components for its activity.

• Connector loop is similar to nuclear cap-binding complex CBC20 N terminal extension (Mazzaet al, 2002) 

Figure 7. Alignment of CBC20 with Cwc2 (CBC in orange and Cwc2 in blue)

There is conservation that is reflected in the amino acid sequence to an extent as well. We can see that the alpha helices are roughly in the similar positions above. This gives further proof that the novel structure of Cwc2 is evolutionarily conserved and therefore advantageous in carrying out its function of RNA binding. The important Y120 residue on cwc2 connector loop is conserved in CBC at tyrosine-20 position on its N-terminal extension. Y20 on CBC could not be visualised above as the CBC80 subunit structure was used. Further work on CBC will allow us greater insight into mechanism of binding. 

Figure 8. Structure of CBC20 subunit with key residues that interact cap highlighted

The yellow residues in Figure 8 are shown in cap-bound conformation whilst the blue residues undergo conformational change to accommodate the cap. The N-terminal extension allows high flexibility much like the connector loop and is key to the functioning of CBC.

• high flexibility, many conformational states ~ important to function as molecular switch (molecular dynamics simulation CHARMM)

The high flexibility of the connector loop can be further characterised in terms of its molecular dynamics. A simulation program such as CHARMM can be run to look at the extent of flexibility the connector loop can accommodate. 

• Molecular switch (accommodate shifting contacts in spliceosomal transitions) 

Figure 9. Ramachandran plot of whole Cwc2 protein (drawn using RAMPAGE)

Figure 10. Ramachandran plot for Connector loop (residues 116-133) using Ramachandran Plot 2.0 program

We see that generally there is much allowed flexibility in terms of torsions and phi/psi angles for the whole molecule and for the connector loop specifically too. However this is a very cursory view and further analysis should be carried out by drawing plots using MATLAB based on PDB data.  However we can tell that the conformations of the loop does not change so quickly that the electron density cloud could not be fixed enough for x-ray crystallography visualization. We can conclude that there are different conformational states associated with the connector loop and that this property might be integral to Cwc2 function.