Transport through the nuclear poresTransport through the nuclear pores

• The NLS and NES consist of short sequences that are necessary and sufficient for proteins to be transported through the nuclear pores.

• Transport receptors have the dual properties of recognizing NLS or NES sequences and binding to the nuclear pore.

• The direction of transport is controlled by the state of the monomeric G protein, Ran.

• The nucleus contains Ran-GTP, which stabilizes export complexes, while the cytosol contains Ran-GDP, which stabilizes import complexes.

• The mechanism of movement does not involve a motor.

Biologia molecolare - Robert F. Weaver Copyright © 2005 – The McGraw-Hill Companies srl

Summary of methods to assess mRNA stability in eukaryotic cellsSummary of methods to assess mRNA stability in eukaryotic cells

Method Advantage Disadvantage Comments

Pulse-chase labeling with 3H-U

measurement of "true" chemical half life

low sensitivityfor high abundance,

slow turnover mRNAs

Injection of in vitro transcribed 32P-

RNA

measurement of "true" chemical half life

lack of cellular RNA modifications, labour

intensive

oocytes can differ from somatic cells,

Inducible promoterrelatively rapid

inductioninduction may alter cell

physiologyHsp70 and myc

promoter

Pharmacological transcription block

can be applied to all genes, rapid onset

block

perturbation of cellular metabolism,

Actinomycin D, and DRB most commonly

used

Comparison of transcription rate and steady state

mRNA level

can be applied to all genes, useful

screening procedure

mRNA stability is not directly measured

should only be used in combination with

another method

In vitro RNA degradation

easy, identification of intermediates,

purification of trans-acting factors

difficult to establish

physiological relevance and

specificity must be established

mRNA degradative activities in mammalian cells

Decapping•DCP2 which binds RNA as a prerequisite for cap recognition. •DCP1 augments DCP2 activity •LSM (SM-LIKE) PROTEINS augment DCP2 activity

5’ -to-3’ exonuclease activity•XRN1 is a proven 5’ -to-3’ exonuclease that localizes to the cytoplasm.•RAT1/XRN2 is only thought to be a 5’ -to-3’ exonuclease on the basis of its similarity to the yeast orthologue.

Deadenylation•PARN is one of five mammalian homologues to yeast Caf1/Pop2 protein

3’ -to-5’ exonuclease activity•Exosome (six RNase-PH-DOMAIN components, PM/SCL75,MTR3,RRP41, RRP42, RRP43 and RRP46; three S1 and KH RNA-binding components,RRP4, RRP40 and CSL4; the RNASE D-like components PM/SCL100; the putative helicaseKIAA0053; and a protein that is phosphorylated in the M phase of the cell)

PMR1-like activity•Polysomal ribonuclease 1 (PMR1) is a polysome-associated mRNA endonuclease

ARE-binding proteinsARE-binding proteinsProtein kDa Motif Expression site ARE Function

AUF1 37,40,42,45 RRM Ubiquitous c-myc, c-fos, GM-CSF mRNA destab.

AUBF ND ND T cells c-fos, INF, IL-3 v-myc, GM-CSF, (AUUUA)n

ARE-binding corr. with mRNA stab.

AU-A 34 ND T cells TNF, GM-CSF, c-myc ND

AU-B 30

AU-C 43

hnRNPA1 36 RRM Human PBMCs GM-CSF, IL-2, c-myc ND

hnRNPC 43

Elav-like 36–40 RRM Ubiquitous, nervous system c-myc, c-fos,TNF-a,GM-CSF mRNA stab.

HuR

HuD

HuC

Hel-N1

TIAR 40, 42 RRM Brain, spleen, lung, liver,testis TNF, GM-CSF Transl. inhib.

TIA-1 Brain, spleen, testis

TTP 44 Cys3His Fibroblasts, macrophages TNF, IL-3 GM-CSF mRNA destab.

KSRP 78 KH Neural cells and other types c-fos mRNA destab.

•AUBF, AU binding factor ; AU-A, AU binding factor-A ; AU-B, AU binding factor-B ; AU-C, AU binding factor-C ; hnRNP, heterogeneous nuclear ribonucleoprotein ; KH, hnRNP-K homology domain; KSRP, KH-type splicing regulatory protein 1; ND, not determined; PBMC, peripheral blood mononuclear cell.

32

Meccanismo dell’ NMD

UAGAUG

mGppp AAAAAAAAAAAAAAAA3 3

2

mGpp p AAAAAAAAAAAAAAAA3

UAG

AAAAAAAAAAAAAAAAmGppp

UAG

3 322 1

RF

RNA interference

• Meccanismo

• Significato biologico

• Strumento di analisi della funzione dei geni

Co-soppressione

Introduzione di copie transgeniche di un gene risulta nella

ridotta espressione del transgene e del gene endogeno

RNA interference

Introduzione di RNA a doppio filamento (dsRNA) induce

silenziamento genico

Componenti dell’RNAi

• siRNA (small interfering RNA) = frammenti di RNA 21-25 nt

• Dicer = endoribonucleasi specifica per dsRNA (tipo RNasi III)

• RISC (RNA-induced silencing complex) = complesso ribonucleoproteico

• RdRP (RNA-dependent RNA polymerase)