Poly A tail

• Turns over (recycles) in cytoplasm• Bound by poly A-binding protein: PAB1 in

cytoplasm, PAB2 in nucleus– Plants have more than 2 PAB genes; some show

tissue-specific expression

• Promotes mRNA stability – When polyA tail too short, mRNA degraded

• Enhances translation– Synergistic stimulation with Cap!

Firefly luciferase mRNA (with plant 5’ and 3’ UTRs) was electroporated into protoplasts (from D. Gallie).

Transgenic tobacco plant expressing the firefly luciferase.

The promoter of the chimeric luc gene is the 35S promoter from Cauliflower Mosaic Virus.

The transgenic plant was sprayed with the substrate (luciferin), placed in darkness and exposed to film.

(From D. Ow and S. Howell)

Glowing Plants!

Common reporter genes for plants1. GUS - E. coli β-glucuronidase

- enzymatic assay (fluorescence product) and histochemical stain assay (in situ), both with synthetic substrates

2. Luc - luciferase from Photinus (firefly), Vibrio (bacterium), Renilla (soft coral)

- assay luminescence (light emission) in vitro or in vivo

3. GFP - green fluorescent protein, intrinsically fluorescent, also from bioluminescent organisms, visualize expression in vivo

- YFP and other engineered colors

GUS staining of a transgenic tobacco plant

Gene is expressed well in root and cotyledon.

Nuclear RNA Splicing

• Two types of introns unique to nucleus:

1. nuclear mRNA introns2. nuclear tRNA introns (Fig. 6.42, Buchanan et al.)

• Splicing mechanisms quite different, but both require proteins

– mRNA splicing may be RNA-catalyzed.

Nuclear mRNA (NmRNA) Splicing

• Nearly all NmRNA introns begin with GU and end with AG

• In yeast & mammals, NmRNA splicing occurs on a spliceosome, which are dynamic ribonucleoproteins (RNPs) composed of several smaller snRNPs and the pre-mRNA

• A snRNP contains a small U-rich RNA (U1,U2,U4,U5 or U6) and >8 proteins (7 are common to all, called Sm proteins)

1. A snurp contains a small, nuclear, U-rich RNA (snRNAs = U1, U2, U4, U5 or U6), and > 8 proteins, 7 (Sm) are common.

2. The snRNAs base-pair with the pre-mRNA (U1, U2, U5, U6) and/or with each other (U4-U6 and U2-U6).

3. Lupus patients have antibodies to snurps; mainly the Sm proteins.

Spliceosomes contain Snurps (snRNPs, small nuclear ribonucleoproteins)

Fig. 14.28Structure of the U1 SnRNP

U1 and U2 paired with pre-mRNA in yeast

The Spliceosome Cycle of Assembly, Rxn, and Disassembly

Fig. 14.22

Similar active sites (catalytic center) in Spliceosomal and Group II introns?

(both models after first step)

Plant NmRNA splicing

1. Plant mRNA introns also follow the GU/AG rule, but tend to be small (< 300 bp)

and very A-U rich in dicots.• A-U rich sequences essential for splicing in

dicots, but can also stimulate splicing in monocots

2. Monocot and dicot snRNPs also differ, and monocot introns spliced poorly in

dicots. 3. Plants have homologues of the snRNAs,

suggestive of a splicing mechanism similar to yeast, but must have some unique factors recognizing the A-U rich regions.

Alternative splicing: regulating photosynthesis

• Can generate 2 (or more) mRNAs, and 2 different proteins, from the same gene

• First case in plants: RuBPCase Activase (Werneke et al. 1989. Plant Cell 1: 815)

– activates RuBPCase– ATPase activity– a chaperonin (protein-specific ?)

Activation by RuBPCase Activase

• Until 1974 only semi-functional RuBPCase could be isolated

• Activase induces folding of inactive RuBPCase• A lysine in the active site is converted to a carbamate

(converts positive charge to negative charge)• Mg2+ coordinates to the carboxyl • “ECM” form of RuBPCase is the real functional unit

R-C-N-H+ + CO2 R-C-N-C = O

H

H

O-2H+

RuBPCase Activase Alternative Splicing (continued)

• Alternative 5' splice sites for the last intron (intron 6) produces 2 forms of the mRNA, and 2 enzyme forms (41 and 43 kDa) with different carboxyl terminal amino acids

• Only the longer form is redox regulated via a disulfide bond between cysteines (S-S).

• Provides for redox regulation of RuBPCase activation.

Alternative splicing involved in regulating flowering via the FCA gene.

FCA promotes flowering, has RNA-binding domain.

4 transcripts from gene, but only the transcript with all 21 introns removed (γ) is functional.

γ only found in certain tissues at certain times (preceding flowering).

Alternative Splicing: Regulating Development

• Arabidopsis Genome Project: ~ 11% of genes alternatively spliced

• Very common among human genes (~ 40%).

Regulation of mRNA stability

• mRNAs generally have limited lifetimes, half-life can be regulated.

• Plant cells have a lot of RNases (particularly in vacuole) that could potentially

degrade mRNA, but which do is not clear. • 3' UTRs and poly-A tails probably important

for stability of most mRNAs, but 5’ UTR could also play a role.

• A few specific instability elements have been identified (AU-rich elements in 3’ UTRs

of certain unstable plant mRNAs).

Hybridization Probes:

CHI, CHS & PAL genes encode enzymes known to be induced by fungal elicitor – produce antibiotics.

H1 contains a rRNA gene (control)

pIBI24 – vector control

(A) Steady-state levels of RNAs

(B) Gene transcription rates as estimated by “run-on transcription in isolated nuclei”

Regulation of Proline-Rich Protein mRNA Stability

Conclusion: ?

(Zhang et al. 1993-94)

Nuclear Run-on Transcription Assay

Effect of protein synthesis inhibitors on PRP mRNA down-regulation (i.e., destabilization) by elicitor (Eli)

Inhibitors:Chx – cycloheximideEme – emetineAni- anisomycin

Ani most effective!

Conclusion: ??

Proline-rich-protein (PRP) mRNA regulation by fungal infection.

1. Gene encodes a cell-wall protein. Physiological reason for PRP mRNA destabilization? Part of the remodeling of the cell wall in response to infection.

2. The protein synthesis requirement for destabilization of PRP mRNA suggests role for an inducible protein.

3. They identified an Instability Element located in the 3’ UTR of PRP mRNA.

4. Also, identified a 50-kD protein that binds to the instability segment: however, already present before induction!

- Maybe the RNA-binding activity of the 50-kD protein regulated by another protein (that responds to elicitor)?

Yeast 2-Hybrid System for identifying interacting proteins

Wikipedia

Gal4 - transcription factor, split into DNA-binding and activation domains.Bait and Library- proteins that interact within the yeast cell.Reporter – lacZ (β-galactosidase) activated when B & L proteins bring Gal4

domains back together.

Translational regulation

• Get global changes in translation

• And differential translation of specific mRNAs

• In vivo mechanisms not well understood in plants; however, in vitro

biochemistry of translation is well studied in wheat germ

Regulation is usually at the level of initiation.

Initiation mechanism differs from prokaryotes and the chloroplast (no S-D sequences, more factors)

Fig. 9.10, Buchanan

Translation overview: 3 stages

Scanning Model of Initiation

• Proposed by M. Kozak• Small subunit of ribosome (+ initiation

factors, GTP and tRNAiMet) binds to the 5’ Cap, and then scans the mRNA until the first

AUG is reached• Translation starts at the first AUG• Translation start site and efficiency also

affected by RNA structure at the 5’ end of the mRNA

Fig. 9.12

Fig. 9.12

eIF-4G may be responsible for the synergism of Cap and polyA tail by interacting with eIF-4E and Pab1p.

Why interact with both Cap and polyA tail?

Initiation factors (except eIF-4)

eIF-1 (& 1A): promotes several steps (modestly)*eIF-2: binds tRNAiMet to 40S subunit, requires GTP

(which gets hydrolyzed to GDP) eIF-2B: catalyzes exchange of GTP for GDP on

eIF-2*eIF-3: binds to 40S subunit, prevents 60S subunit

from binding to iteIF-5: stimulates 60S subunit binding to the 40S

pre-initiation complex*eIF-6: binds to 60S subunit, helps prevent 40S

subunit binding

eIF-4

eIF-4F: Cap-binding complex, composed of 3 subunits eIF-4E, eIF-4A, & eIF-4G

(eIF-4E binds directly to Cap)

eIF-4A: RNA helicase, DEAD motif

eIF-4B: binds RNA, stimulates eIF-4A

eIF-4G: versatile adaptor protein,interacts w/eIF-4E, eIF-3, and poly-A binding protein (Pab1p)