An active precursor in assembly of yeast nuclear ribonuclease P

Abstract

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.

Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP

“…Pre-RPR1 RNA is assembled into a ribonucleoprotein complex with at least seven protein subunits of RNase P, including Pop1p, before being processed to the mature RNA (Srisawat et al 2002). Mutations in RPR1 RNA that compromise its structure or prevent protein association also slow maturation of the RPR1 RNA, possibly by blocking the formation of the correct RNP complex required for processing.…”

“…RPR1 RNA is synthesized as a precursor (pre-RPR1 RNA) that contains an 84-nucleotide (nt) 59-leader sequence and a 39-trailing sequence flanking the mature RPR1 sequence (Lee et al 1991a). Pre-RPR1 RNA is assembled into a ribonucleoprotein complex with at least seven of the nine RNase P protein subunits before being processed to the mature form (Srisawat et al 2002).…”

“…Pop1p is important for the assembly and cellular functions of RNase P and MRP (Lygerou et al 1994;Srisawat et al 2002), and is conserved from yeast to human (Lygerou et al 1996b). Attempts to characterize the function of Pop1p in vitro have not been successful, largely because the individually expressed protein is not soluble and the yeast holoenzyme has not been reconstituted in vitro.…”

Characterization of conserved sequence elements in eukaryotic RNase P RNA reveals roles in holoenzyme assembly and tRNA processing

“…Since the pre-RPR1 RNA binds at least seven of nine protein subunits before maturation (Pop1p, Pop4p, Pop5p, Pop6p, Pop7p, Pop8p, and Rpp1p) (Srisawat et al 2002), defects in the RNA structure could alter the rate of precursor processing either directly or indirectly through blocking some essential RNP formation. Depletion of any of the seven protein subunits in the precursor complex, or compromised RNA structure or protein binding, will block maturation (Lygerou et al 1994;Pagan-Ramos et al 1996a,b;Chu et al 1997;Stolc and Altman 1997;Chamberlain et al 1998;Ziehler et al 2001).…”

“…The RPR1 RNA is synthesized as a precursor form, pre-RPR1 RNA, which contains an 84-nt 5 0 leader, where an internal promoter element for RNA polymerase III resides, and a short 3 0 -trailing sequence (Lee et al 1991a). In actively growing yeast culture, the pre-RPR1 RNA appears to be converted to the mature form by removal of the 5 0 -leader and 3 0 -trailing sequences after seven of the nine protein subunits have associated with the RNA (Srisawat et al 2002), and the ratio of mature to precursor RPR1 RNA is $5:1.…”