Clp1, an overexpressed subunit of CF IA and insights into its molecular role using biophysical approaches

Abstract

In eukaryotes, pre-messenger RNA (pre-mRNA) 3′-end processing is vital for gene expression and mRNA biogenesis; any awry in this pre-mRNA processing has detrimental effects and causes various diseases in humans. Pre-mRNA processing is coordinated via complex multi-protein machinery involving over 20 protein factors in Saccharomyces cerevisiae. Endonucleolytic cleavage at the pre-mRNA 3′-end followed by poly(A) tail addition at the newly generated 3′-end requires CF IA, CPF, CF IB and other sequence elements. Among these essential proteins, Clp1 of the CF IA sub-unit is indispensable for the effective functioning of 3′-end processing machinery. Clp1, a conserved protein, is proposed to interlink 3′-end processing, transcriptional elongation and transcription termination. However, experimental evidence defining the precise roles played by Clp1, its interacting partners from other subunits and information about its exact mechanism of action during pre-mRNA processing is lacking. The current study details on the Clp1 N-terminal domain's (1–100 amino acids referred to as Clp11–100) successful cloning, expression and characterization using numerous biophysical techniques like circular dichroism and mass spectrometry etc. This information would open new frontiers to decipher the structure and molecular role of Clp1 during pre-mRNA maturation.