Transport 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.
Summary of methods to assess mRNA stability in eukaryotic cells MethodAdvantageDisadvantageComments Pulse-chase labeling with 3H-U measurement of "true" chemical half life low sensitivity for 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 promoter relatively rapid induction induction may alter cell physiology Hsp70 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 Deadenylation PARN CCR4/CAF1/NOT complex PAN2/PAN3 complex 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. 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
Elementi di regolazione della stabilità dell'mRNA