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, 2003-2019, 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), 5408–5416

3.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 : 32P-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

PNPase Rrp41 Rrp41p/Ski6p hRrp41p RNase PH Q17533

(x 3) (x 3) Rrp46p hRrp46p RNase PH (Crn-5)

Mtr3p hMtr3p RNase PH

Rrp42 Rrp42p hRrp42p RNase PH NP_508024

(x 3) Rrp43p OIP2 RNase PH

Rrp45p PM/Scl-75 RNase PH T28842

Csl4 Csl4p/Ski4p hCsl4p S1 RBD

Rrp4p Rrp4p hRrp4p S1/KH

Rrp40p hRrp40p S1/KH

Other common proteins:

(RNase R?) Rrp44p/Dis3p (hDis3p) RNase R

(RNase D?) Rrp6p PM/Scl-100 RNase 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.