Lecture 7: RNA processing and interference

3 steps in eukaryotic mRNA processing

5’ end capping- methyl guanosine is added,

3’ end polyadenylation- addition of poly A tail,

Splicing- noncoding introns are removed.

mRNA definition

RNA that will be translated into protein. Has 5’ cap and 3’ poly A sequence. Has coding and noncoding regions. Starts as primary transcript in nucleus and is modified and exported to the cytoplasm.

Co-transcriptional processes

RNA processing steps are coupled to transcription

All associate with the carboxy-terminal domain of RNA polymerase 2

5’ end capping factors, polyadenylation/3’ end cleavage factors, sliceosome

CTD of RNA polymerase 2

C-terminal Domain, serves as scaffold for organization of pre-mRNA processing factors

Functions of 5’ 7-methyl Guanosine cap on mRNAs

prevents degredation at 5’ end by exonucleases, assists in moving the mRNA out of the nucleus, serves as recognition site for initiation of translation, helps in splicing.

Purpose of the Poly A tail

regulated mRNA stability and prevents degradation of 3’ end by exonucleases, Regulated nuclear exports, utilized in protein translation, useful to scientists for isolating mRNAs by hybridization with poly dT column.

3’ end of the mRNA processing

poly a recognition site is encoded in RNA, functions as recognition site for assembly of 3’ end processing complex, 3’ end of primary transcript is cleaved by exonuclease (~20nt downstream of poly A), poly A is added after the cut site.

Poly A recognition site

AAUAAA

Poly A polymerase (PAP)

Adds poly A tail

Addition of PolyA tail

CPSF cleaves primary transcript to shorten 3’ UTR, PAP adds poly A tail, Poly A binding proteins bind tail, additional poly A binding proteins added in cytoplasm.

CPSF

Cleavage and Polyadenylation Specificity Factor- cleaves primary transcript to shorten 3’ UTR

Up to __ % of genes are alternatively spliced

90

__% of inherited diseases result from incorrect splicing

15

Introns are processes from ____.

pre-RNA

RNA splicing

allows multiple mRNAs and multiple proteins to be produced from a single gene. Process must occur precisely.

RNA splicing is coordinated by a protein/ small nuclear complex (____) called a ____.

snRNA; spliceosome

The average human gene contains __ introns (comprising __% of the transcription unit.

9; 90

Exons

Average size in humans is 150 nt; always recombined in the same linear order

Introns

average size in humans is 3500 nt

pre-mRNA

same sequence as DNA, matches the DNA sequence of the 5’ strand. Uses U’s instead of T’s. Multiple mRNAs can be produced from pre-mRNA by different combinations of exons. has unique combination of exons.

Spliceosome

recognizes specific sequences in the intron, introns are removed by formation of lariat and ligation of the 2 adjoining exons.

Small nuclear RNAs

required for splicing.

U1snRNP

binds to conserved 5’ splice site

U2AF1snRNP

binds to conserved 3’ splice site

snRNP

facilitates 2 transesterification events to remove intron as a lariat

RNA interference

Method of down-regulating gene expression, regulation occurs through binding of an RNA to another RNA molecule and targeting the RNA for degradation or preventing translation. Also known as RNA silencing

siRNA

Small interfering RNA, short (20-25nt) double stranded RNA molecules, Exogenous to mammalian cells, have short (2-3) bp overhang on 3’-OH end. Interferes with expression of specific genes to prevent synthesis of a protein. (targets mRNA for degradation or prevents translation of mRNA)

Dicers

Double stranded RNA from viruses can be cleaved by dicer to make siRNA in lower eukaryotic organisms. Eukaryotic organisms also recognize siRNAs that don't require processing by Dicer. Endoribonuclease that can cut a ds DNA to make an siRNA

siRNA

Small interfering RNAs, small ds rnas that can target mRNAs for degradation, inhibit translation of mRNA into protein. Dicer is endoribonuclease that can cut a ds DNA to make an siRNA

siRNAs target ___ for degradation

mRNAs

Small interfering RNAs

Mechanism for reducing protein levels through RNA regulation. Mech. is the same for miRNA after initial steps

miRNA

microRNA, endogenous evolutionarily conserved non coding RNA that regulate gene expression. 20-23nt ds non coding RNAs that are encoded into genome. can function when sequences are not 100% match with target mRNA. typically regulates multiple genes.

miRNA function

Decrease gene expression by inactivating mRNA to reduce protein levels

miRNA can be encoded with___

introns, within the antisense orientation within a gene or in intergenic region.

miRNAs are transcribed by

RNA Pol 2

Endogenous

mechanism for interfering with protein production

pri-miRNA

processed into miRNAs

DROSHA

cleaves pri-miRNA to make pre-miRNA

1

Pre-miRNAs are exported out of the nucleus

2

Pre-miRNA is cleaved by Dicer in cytoplasm to make an miRNA

miRNAs can block translation of mRNAs

miRNA + argonaut

pre-RISC complex

Argonaut

Removes passenger strand of DNA

If guide RNA is not a perfect match

No translation occurs; Typical for miRNA

If guide RNA is a perfect match

it targets mRNA for degradation

Long noncoding RNA (lncRNA)

200nts, can be transcriptional units or transcribed from within enhancers, promotors, introns or pseudogenes

lncRNA functions

occurs in many different cellular processes but it’s still poorly understood

piRNAs function

small RNAs that suppress the movement of transposable elements in the germline.

piRNAs

piwi-interacting RNAs, longer than other small RNAs- 24-32 nt. Interact with piwi proteins to silence mobile genes known as transposons and can participate in the RISC complex to participate in gene sliencing.

formation of piRNAs

does not involve the formation of dsRNA precursors or cleavage by the Dicer.

A

Poly-A polymerase (PAP)

B

Poly-A binding protein (PABP)

C

Cleavage and Polyadenylation Specificity Factor (CPSF)

E

3’ end