Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins The Plant Cell, Vol. 15, , September 2003 Shlomit Yehudai-Resheffa, Victoria Portnoya, Sivan Yogeva, Noam Adirb and Gadi Schuster1 Mike 7/14

The function of spinach chloroplast polynucleotide phosphorylase (PNPase) 1.chloroplast mRNA degradation (chloroplast and bacteria) 1). endonucleolytic cleavage 2). addition of poly(A)-rich sequences to the endonucleolytic cleavage products (PAP in E.coli) 3). exonucleolytic degradation 2.PNPase in the chloroplast was found to form a homotrimeric complex and lacks any known interactions with other proteins. RNA. 7, (2001), 1464–1475 Mol. Cell. Biol. 21, (2001), 5408– No PAP can be detected in spinach chloroplasts, and thus both polyadenylation and degradation are performed by one enzyme, PNPase

The spinach chloroplast PNPase structure is similar to that of the bacterial enzyme The amino acid sequence and domain structure is largely conserved between bacteria and organelles. core = RNase PH domain

RNA degradation and polyadenylation activities of the spinach chloroplast PNPase and its domains # bacteria : domain 2 have activity only Degradation : domain 1 and 2 Polymerization : domain 2 Product : NDP (TLC)

The high-affinity poly(A) binding site is located in the S1 Domain UV light cross-linking assay (Lisitsky et al., 1997b; Lisitsky and Schuster, 1999). UV light cross-linking competition assay substrate : 32 P-psbA RNA domain 2 (only)

Unlike the FL PNPase, the proteins that include only one core domain do not pause at a stem-loop structure PNPase enzyme is its pausing at a stem-loop structure when processively degrading RNA. EMBO J. (1996) 15, 1132–1141

A platform of 6 to 12 nucleotides 3' to the stem loop is required for RNA polyadenylation by PNPase E. coli PAP I is inhibited by a stem-loop structure but that the addition of two nucleotides 3' to the stem loop is sufficient to promote efficient polyadenylation. Nucleic Acids Res.(2000) 28, 1139–1144.

The spinach chloroplast PNPase and its active fragments complement the growth of an E. coli PNPase- and RNase PH–less Strain at 18°C E. coli strain SK 8992

Summary

Exosome Core Proteins: 6 x RNase PH + 3 x S1/KH E. Coli Archeal Yeast Human PNPaseRrp41Rrp41p/Ski6phRrp41pRNase PH Q17533 (x 3) (x 3)Rrp46phRrp46pRNase PH (Crn-5) Mtr3phMtr3pRNase PH Rrp42Rrp42phRrp42pRNase PH NP_ (x 3)Rrp43pOIP2RNase PH Rrp45pPM/Scl-75RNase PH T28842 Csl4Csl4p/Ski4phCsl4pS1 RBD Rrp4pRrp4phRrp4pS1/KH Rrp40phRrp40pS1/KH Other common proteins: (RNase R?)Rrp44p/Dis3p (hDis3p)RNase R (RNase D?)Rrp6pPM/Scl-100RNase D (Crn-3) (nuclear only) Chloroplast PNPase (x3 ?) domain 2 domain 1 red: in vitro 3’-5’exonuclease activity PNPase lacks any known interactions with other proteins.