RNA Polymerase II (Pol II) transcripts comprise many different RNA classes that undergo co-transcriptionally complex maturation processes including the addition of a 5’ cap structure, removal of introns via splicing, 3’ end processing and the assembly into transport competent ribonucleoprotein particles. How all these processes are coordinated, regulated and quality controlled, is a central question in RNA biology. One of the processes all Pol II transcripts have in common is the addition of a cap structure to the 5’ end. This 5’ cap structure is immediately bound by the heterodimeric cap binding complex CBC comprising subunit CBP80 and the cap binding subunit CBP20. The cap structure as well as CBC are... More
RNA Polymerase II (Pol II) transcripts comprise many different RNA classes that undergo co-transcriptionally complex maturation processes including the addition of a 5’ cap structure, removal of introns via splicing, 3’ end processing and the assembly into transport competent ribonucleoprotein particles. How all these processes are coordinated, regulated and quality controlled, is a central question in RNA biology. One of the processes all Pol II transcripts have in common is the addition of a cap structure to the 5’ end. This 5’ cap structure is immediately bound by the heterodimeric cap binding complex CBC comprising subunit CBP80 and the cap binding subunit CBP20. The cap structure as well as CBC are crucial for the correct processing of the nascent transcript. The subsequent maturation processes including the involved processing machineries depend on the RNA class. However, some proteins like NELF-E (negative elongation factor subunit E) and ARS2 (arsenite-resistance protein 2) seem to associate with the RNA-CBC complex regardless of the type of RNA, whereas other CBC interacting proteins like the phosphorylated adaptor for RNA export PHAX are important for distinct RNAs. To better understand the individual CBC complexes, different biochemical, biophysical and structural methods were used to analyse the individual interactions between CBC and ARS2, PHAX as well as NELF-E on molecular level. The studies showed the incompatibility of ARS2 and NELF-E binding to CBC and revealed that both proteins are sharing the same binding site on CBC and that their interacting residues show high conservation and similarities. In addition, solving the structure of human ARS2 allowed the identification of domains and residues involved in RNA and protein-protein interactions. Further analysis also led to the identification of a novel CBC-ARS2 complex, including the nuclear cap binding protein NCBP3, namely CBC-ARS2-NCBP3. The results obtained support the hypothesis that ARS2 serves as additional binding surface for proper sorting and processing of nascent transcripts. Additionally, all results together enable to propose a model of RNA processing containing mutually exclusive RNA-CBC complexes based on the RNA maturation stage as well as the RNA class.
