Regulación Post-transcripcional en eucariotas Biología Molecular

Regulación Post-transcripcional
en eucariotas
Biología Molecular
2009
Figure 7-92 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-104 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-105 Molecular Biology of the Cell (© Garland Science 2008)
Figure 7-106 Molecular Biology of the Cell (© Garland Science 2008)
Wilusz y Wilusz TiGs 2004
Parker y Song, Nature Rev SMB 2004
Wilusz et al, Nature Reviews MCB 2001
Wilusz y Wilusz TiGs 2004
Exosomes
February 2007 Molecule of the Month
http://www.wwpdb.org
Wilusz y Wilusz TiGs 2004
Wilusz y Wilusz TiGs 2004
Nuclear mRNA decay
Figure 7-111 Molecular Biology of the Cell (© Garland Science 2008)
Nonsense-mediated mRNA decay
(NGD)
Modificado de Mata et al TIBS 2005
Parker y Song, Nature Rev SMB 2004
Parker y Song, Nature Rev SMB 2004
Wilusz y Wilusz TiGs 2004
Figure 1General scheme of messenger RNA decay pathways. (A)
The regulation of gene expression involves the control of mRNA
degradation at the post-transcriptional level. mRNAs containing an
AU-rich element (ARE) in their 3' untranslated region (UTR) undergo
rapid ARE-mediated mRNA decay (AMD) in resting cells. Stabilization
of ARE-containing mRNAs by various stimuli contributes to the
induction of gene expression. (B) Quality control mechanisms activate
mRNA degradation. mRNAs that contain a premature termination
codon (PTC) are recognized and specifically degraded by the
nonsense-mediated mRNA decay (NMD) pathway. (C) The basic
mRNA decay machinery in the cytoplasm initially removes the poly(A)
tail through the activity of deadenylating enzymes. Subsequently, the
mRNA can be further degraded from the 3' end by a complex of 3'–5'
exonucleases known as the exosome. Alternatively, the mRNA is
decapped at the 5' end, and the 5'–3' exonuclease Xrn1 proceeds to
Newbury et al. EMBO reports 7, 2, 143–148 (2006)
doi:10.1038/sj.embor.7400628
Micro ARNs
Son ARN no codificantes
• Pequeños
• Regulatorios
• Endógenos
Intergénica: tendencia a agruparse en clusters
– Transcritos primarios policistronicos
– Promotores ARN PolII, (splicing), 5’-cap, y cola
poliA
Intrónica: mayormente en orientacion sentido
– Pueden estar en clusters
– Por lo menos algunos transcritos por ARN PolII
Figure 7-112 Molecular Biology of the Cell (© Garland Science 2008)
Sintesis y procesamiento de los microRNAs:
•
1. Transcipcion
miRNAs are initially expressed as part of transcripts termed primary miRNAs
(pri-miRNAs) (Lee 2002). They are apparently transcribed by RNA Polymerase
II, and include 5' caps and 3' poly(A) tails (Smalheiser 2003, Cai 2004). The
miRNA portion of the pri-miRNA transcript likely forms a hairpin with signals for
dsRNA-specific nuclease cleavage.
•
2. Remocion de la horquilla en el nucleo DROSHA
The dsRNA-specific ribonuclease Drosha digests the pri-miRNA in the nucleus
to release hairpin, precursor miRNA (pre-miRNA) (Lee 2003). Pre-miRNAs
appear to be approximately 70 nt RNAs with 1–4 nt 3' overhangs, 25–30 bp
stems, and relatively small loops. Drosha also generates either the 5' or 3' end
of the mature miRNA, depending on which strand of the pre-miRNA is selected
by RISC (Lee 2003, Yi 2003).
•
3. Exportacion al citoplasma
Exportin-5 (Exp5) seems to be responsible for export of pre-miRNAs from the
nucleus to the cytoplasm. Exp5 has been shown to bind directly and specifically
to correctly processed pre-miRNAs. It is required for miRNA biogenesis, with a
probable role in coordination of nuclear and cytoplasmic processing steps.
(Lund 2003, Yi 2003).
4. Procesamiento por DICER
Dicer is a member of the RNase III superfamily of bidentate nucleases that has
been implicated in RNA interference in nematodes, insects, and plants. Once in
the cytoplasm, Dicer cleaves the pre-miRNA approximately 19 bp from the
Drosha cut site (Lee 2003, Yi 2003). The resulting double-stranded RNA has 1–
4 nt 3' overhangs at either end (Lund 2003). Only one of the two strands is the
mature miRNA; some mature miRNAs derive from the leading strand of the primiRNA transcript, and with other miRNAs the lagging strand is the mature
miRNA.
5. Seleccion de hebra por RISC
To control the translation of target mRNAs, the double-stranded RNA produced
by Dicer must strand separate, and the single-stranded mature miRNA must
associate with the RISC (Hutvagner 2002). Selection of the active strand from
the dsRNA appears to be based primarily on the stability of the termini of the
•
•
Figure 7-113 Molecular Biology of the Cell (© Garland Science 2008)
Interferencia de ARN
•Es el proceso por el cual la introducción de ARN doble hebra en una célula causa la
degradación específica de los ARNm que contienen la misma secuencia.
•Funciona en C. elegans
Fire et al. Nature 1998
Knock out:
hebra antisentido y sentido
tenían efecto semejante
la mezcla de ambas hebras
logra un efecto 10x antisentido
Carlo Cogoni Giuseppe Macino
Figure 7-115 Molecular Biology of the Cell (© Garland Science 2008)
GFP-expressing worms….
RNAi by ingesting dsRNA!
Timmons and Fire
Nature 1998
295, 854
Bacteria expressing
dsRNA
….eating regular
bacteria
….eating bacteria expressing
GFP dsRNA
Comparacion de microRNA y siRNA
• localización del ARNm
• estabilidad del ARNm
• vigilancia de la calidad del ARNm
• micro ARNs / interferencia
• regulones de ARN