NAF1 and SHQ1 are involved in early events of box H/ACA RNP biogenesis

Abstract

H/ACA ribonucleoproteins are small complexes of four conserved proteins and a small function defining RNA called the H/ACA RNA. Depending on the specific H/ACA RNA (out of the cellular pool of about 150 H/ACA RNAs) that is part of the complex, the H/ACA RNP can modify ribosomal RNAs or spliceosomal RNAs by converting uridines to pseudouridines, process rRNAs, and even play a role in stabilizing chromosome ends. Although consisting of only five components, namely the four proteins NAP57, NHP2, NOP10 and GAR1 and a small RNA, the mechanism of biogenesis of H/ACA snoRNPs is not completely understood. It is understood from studies in lower organisms that assembly factors might be required to aid in the biogenesis process and facilitate the assembly of the complex and the stabilization of the H/ACA RNA. We investigated the role of two putative assembly factors, NAF1 and SHQ1, in the assembly of H/ACA RNPs using a variety of biochemical and cell biological techniques. Both NAF1 and SHQ1 seem to be required for the stability of newly synthesized H/ACA RNA, implicating these two proteins in the biogenesis of H/ACA RNPs. Both proteins are also required to assemble functional particles in an in vitro RNP reconstitution system. Additional proof for the involvement of these proteins in the biogenesis of H/ACA RNPs comes from biochemical interactions among these proteins. Both NAF1 and SHQ1 bind to the H/ACA protein NAP57. In addition, biochemical data for these proteins indicate a series of competitions between the two assembly factors and also a core H/ACA protein GAR1 and implicates an order of assembly for H/ACA RNPs. SHQ1 seems to be recruited at a very early step in assembly and is later replaced by NAF1. NAF1 gives way to GAR1 to form the mature particle. This stepwise assembly may be a way to keep the RNP in an inactive form throughout the cell and release the mature forms only at the site of function. H/ACA ribonucleoproteins are small complexes of four conserved proteins and a small function defining RNA called the H/ACA RNA. Depending on the specific H/ACA RNA (out of the cellular pool of about 150 H/ACA RNAs) that is part of the complex, the H/ACA RNP can modify ribosomal RNAs or spliceosomal RNAs by converting uridines to pseudouridines, process rRNAs, and even play a role in stabilizing chromosome ends. Although consisting of only five components, namely the four proteins NAP57, NHP2, NOP10 and GAR1 and a small RNA, the mechanism of biogenesis of H/ACA snoRNPs is not completely understood. It is understood from studies in lower organisms that assembly factors might be required to aid in the biogenesis process and facilitate the assembly of the complex and the stabilization of the H/ACA RNA. We investigated the role of two putative assembly factors, NAF1 and SHQ1, in the assembly of H/ACA RNPs using a variety of biochemical and cell biological techniques. Both NAF1 and SHQ1 seem to be required for the stability of newly synthesized H/ACA RNA, implicating these two proteins in the biogenesis of H/ACA RNPs. Both proteins are also required to assemble functional particles in an in vitro RNP reconstitution system. Additional proof for the involvement of these proteins in the biogenesis of H/ACA RNPs comes from biochemical interactions among these proteins. Both NAF1 and SHQ1 bind to the H/ACA protein NAP57. In addition, biochemical data for these proteins indicate a series of competitions between the two assembly factors and also a core H/ACA protein GAR1 and implicates an order of assembly for H/ACA RNPs. SHQ1 seems to be recruited at a very early step in assembly and is later replaced by NAF1. NAF1 gives way to GAR1 to form the mature particle. This stepwise assembly may be a way to keep the RNP in an inactive form throughout the cell and release the mature forms only at the site of function.