Regulación Post-transcripcionalen eucariotas

Biología Molecular2009

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

ExosomesFebruary 2007 Molecule of the Monthhttp://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)(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

Newbury et al. EMBO reports 7, 2, 143–148 (2006)doi:10.1038/sj.embor.7400628

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

Micro ARNs

Son ARN no codificantes• Pequeños• Regulatorios• EndógenosIntergénica: 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 DROSHAThe 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 citoplasmaExportin-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 DICERDicer 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 pri-miRNA transcript, and with other miRNAs the lagging strand is the mature miRNA.

• 5. Seleccion de hebra por RISCTo 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)

Bacteria expressing dsRNA

GFP-expressing worms….

….eating regular bacteria

….eating bacteria expressing GFP dsRNA

RNAi by ingesting dsRNA! Timmons and Fire Nature 1998295, 854

Comparacion de microRNA y siRNA

• localización del ARNm

• estabilidad del ARNm

• vigilancia de la calidad del ARNm

• micro ARNs / interferencia

• regulones de ARN