GE-T3-Zhang- Posttranscriptional RNA processing

Exportin5

How is miRNA generated? Exports pre-miRNA

hypermethylated 5' terminal (7mg); 5'-5' phosphate linkage in structure; stabilizing properties

Read properties of Cap structures

nuclear processing of pre-mRNA; transport to the cytoplasm; translation; cytoplasmic mRNA stability

Role of Poly(A) Tail:

poly(a) tail; 3' UTR; mRNA coding region; 5'-UTR and CAP;

Sequence elements for the poly(a) tail:

DNA template; mRNA in the nucleus after transcription

The poly(A) tail is not coded in the ______, but is added to the _______.

rna polymerase; 5' capping enzymes; 3' poly(a) addition enzymes; spliceosome

What are the key proteins involved in mRNA transcription?

RNAP binding site/promoter (start); termination site (end)

Where does transcription start or end on a template DNA?

RNA binding proteins · Poly(A)-binding protein (PABP) · Proteins that bind to AU-rich regions · Iron regulatory protein · Ribonucleotide reductase mRNA-binding protein Trans-acting factors not known to bind RNA · Beta-tubulin, histone, heat shock proteins · P27 rex of human T-cell leukemia virus · Virion host shutoff protein of herpes simplex virus · Regulatory factors affected by translation inhibitors

mRNA STABILITY: Questions to be addressed: Which trans-acting factors regulate mRNA stability and how do they function?

miRNA

micro RNA that regulates gene expression

· Polymerase II transcribes pre-miRNA · Drosha generates pre-miRNA · Exportin5 exports pre-miRNA · Dicer generates a miRNA duplex · Ago loads the guide strand of miRNA onto RISC (RNA-induced silencing complex)

microRNA and small interfering RNA: questions to be addressed: How is miRNA/siRNA generated?

7mG; 5'-penultimate base

All viral mRNAs(w/exceptions) contain _____ on the capping base, and the ______ is always a purine.

a stretch of A residues (≈200 A residues) at the 3'-end; mRNAs encoding histone proteins and some viral mRNAs

Almost all eukaryotic mRNAs have _____, with the exception of _____

protein synthesis; mrna stability; mrna biosynthesis

Biological Role of the Cap Structure

Since the capping and transcription are tightly coupled, it is not clear whether cap formation is involved in, or required for, the initiation of mRNA synthesis. In several viral systems such as vesicular stomatitis virus (VSV), capping may be essential for the synthesis of mRNAs.

Biological Role of the Cap Structure: How is it involved in mRNA biosynthesis?

Cap protects mRNA from degradation by nucleases; The cap structure stabilizes mRNA both in vivo and in cell-free extracts

Biological Role of the Cap Structure: How is it involved in mRNA stability?

Uncapped or unmethylated mRNAs are poor templates for protein synthesis; 7-methylguanosine facilitates binding of mRNAs to 40S ribosome; Chemical analogs of cap, inhibit translation and ribosome binding of capped mRNA; Cap-binding protein has a high affinity to bind the cap and stimulates protein synthesis

Biological Role of the Cap Structure: How is it involved in protein synthesis?

5'-terminus; hyper-methylated guanosine residue (2,2,7-trimethylguanosine); 2'-O-pyrophosphate (PPi) linkage

Capping: When discovered, the ____ end of the RNA was found to contain a ________ linked to a ______

-Small RNA, 19-25 nts in length, 5'-U---, 2 nt 3'-over- hang, precisely excised from dsRNA precursor. -Found in plant (120), invertebrate (150) and vertebrate animals (>2000 in human) -Abundant: up to 10,000 copies/cell

Describe miRNA:

-Small RNA, 21-24 nts in length, 2 nt 3'-overhang, randomly excised from dsRNA precursor. -Endogenous siRNAs are found in plant, but not in mammals or insects.

Describe siRNA:

protein synthesis; mrna stability; mrna biosynthesis

Emphasis: -What biological roles do the cap play in gene expression?

nuclear processing of pre-mRNA; transport of pre-mRNA to the cytoplasm; translation; cytoplasmic mrna stability

Emphasis: -What biological roles do the poly(A) tail play key roles in in gene expression?

Tobacco nucleotide pyrophosphatase; critical for cloning and analyzing mRNA through RACE (Rapid Amplification of cDNA Ends)

Enzyme that cleaves the cap structure and its implications?

RNA polymerase; Nucleotide phosphohydrolase; Guanylyltransferase; Methyltransferases I and II

Enzymes Involved in the Capping Reaction:

exoribonucleases; endoribonucleases;

Enzymes involved in degrading mRNA:

The presence of the poly(A) tail has a practical implication. The poly(A) tail of mRNA base pairs with oligo (U) or oligo (dT), and this reaction can be used to isolated poly(A)+ mRNA. Now commercially available is the oligo (dT) column for isolating poly(A)+ mRNAs.

Explain application of experimentally isolating the poly(a) tail

histone H3 mRNA

Figure. 3'-processing of non-poly (A) mRNA (________)

· The rxn for polyadenylation is catalyzed by Poly(A) polymerase · The nascent mRNA transcript is cleaved by a nuclease at a site upstream of the 3' end · The cleavage process is directed by sequence elements called polyadenylation signals o AAUAAA sequence o Downstream elements o Cleavage site o Upstream sequences o Promoter influence · This newly cleaved 3' end receives the poly(A) tail

How does the polyadenylation take place?

transcription of pri-miRNA (pol II) generation of pre-miRNA (Drosha) exporting of pre-miRNA (exportin5) generation of miRNA duplex (Dicer) loading of the guide strand of miRNA (onto RISC by Ago)

How is miRNA generated? Describe the processes involved

Dicer

How is miRNA generated? Generates miRNA duplex

Drosha

How is miRNA generated? Generates pre-miRNA

RISC by Ago

How is miRNA generated? Guide strand of miRNA is loaded onto _______

pol II

How is miRNA generated? Transcribes pri-miRNA

Nucleotide phosphohydrolase removes a phosphate from the nascent RNA transcript. Guanylyltransferase then adds a Gppp group to the transcript and 2 phosphates are lost leaving a Gp. Methyltransferases I and II then methylate Gp to yield a 7mGP5' (7-methylguanosine) cap.

How is the cap added to the 5'-end of a RNA transcript?

First, transcription is controlled by limiting the amount of mRNA that is produced from a particular gene. The second level of control is through post-transcriptional events that regulate the translation of mRNA into proteins. Even after a protein is made, post-translational modifications can affect its activity.

How is transcription regulated?

200 A residues typically

How long of the poly(A) tail does a typical mRNA have?

universally (including yeasts, molds, plants, insects, and higher organisms,with few exceptions.)

How present are cap structures in eukaryotic mRNAs?

no; Transcripts have to be cleaved by a nuclease at a site upstream of the 3'-end of the nascent transcript; The newly cleaved 3'-end is then receiving the poly(A) tail.

Is the poly(a) tail added to the last nucleotide of the nascent transcript? Why or why not?

Cap 0 (7mGpppN1pN2p-); Cap 1 (7mGpppN1mpN2p-); Cap 2 (7mGpppN1mpN2mp)

Name and describe the three types of caps:

Types of caps are distinguished by how many of the methylation sites have occurred. I. Cap 0: a cap that possesses a single methyl group at the 7 position of the terminal guanine is called cap 0. Cap 0 exists in all eukaryotes with the exception of some viral mRNAs and is the first methylation site of the cap structure. Cap 0 denotes 7mGpppN1pN2p..., where N1 and N2 are purine or pyrimidine ribonucleosides. The enzyme responsible for this modification is called guanine-7-methyltransferase (see below). II. Cap 1: a cap with two methyl groups [one at the 7 position as in cap0, and the other at the 2'-O position of the penultimate base (which is actually the original first base of the transcript before any modifications are made)], described as 7mGpppN1mpN2p..., and exists predominantly in all eukaryotes except unicellular organisms. The enzyme responsible for adding the second methyl group is called 2'-O-methyl-transferase (see below). III. Cap 2: a cap with a third methyl group at the 2'-O position of the third base of the capped mRNA (or the second base of the nascent transcript) is called cap 2, usually described as 7mGpppN1mpN2mp.... This cap usually represents less than 10-15% of the total capped population.

Notice differences between the three types of caps

AAUAAA sequence; downstream elements; cleavage site; upstream sequences; influence of promoters

Polyadenylation Signals:

-Capping base is universally 7-methyl guanosine (7mG). -The unique 5'-5' phosphate linkage results in the retention of a 2', 3'-cis-diol on the 5'-terminal 7mG. -The long triphosphate bridge allows 7mG to bend and intercalate between the bases, thus stabilizing the 5'-terminus of mRNA and allowing interaction with proteins in ribosomes.

Properties of the 5'-cap structure:

cloning and RACE techniques

Quick explanation on experimental method to identify the 5' cap

· The rxn for polyadenylation is catalyzed by Poly(A) polymerase · The nascent mRNA transcript is cleaved by a nuclease at a site upstream of the 3' end · The cleavage process is directed by sequence elements called polyadenylation signals o AAUAAA sequence o Downstream elements o Cleavage site o Upstream sequences o Promoter influence · This newly cleaved 3' end receives the poly(A) tail

Quickly describe process of polyadenylation signals on adding the pol(a) tail

Polyadenylation Signals

The cleavage is directed by sequence elements--_______.

Poly(A) polymerase; free 3'-OH

The polya tail is catalyzed by the template-independent enzyme _______, which adds the poly(A) to the ______ end of an mRNA.

RNA-binding proteins; Trans-acting factors not known to bind to RNA

Trans-acting regulatory factors of poly(a) tail:

histone mRNA 3'-terminal stem-loop, AUREs, IREs, Long-range stem-loop of insulin-like growth factor II (IGF-II)

Types of 3'UTR sequences in the poly(a)

· Cytoplasmic mRNA stability · Nuclear processing of pre-mRNA · Translation · Transport of mRNA to the cytoplasm- protects it from degradation

What are the biological roles of the poly(A) tail?

-siRNA is considered exogenous double-stranded RNA that is taken up by cells or enters via vectors like viruses while miRNA is single-stranded and comes from endogenous, non-coding RNA. -In animals, miRNA tends to have a more perfectly complimentary sequence which induces mRNA cleavage as opposed to just repression of translation.

What are the differences between miRNA and siRNA?

-Both siRNA and miRNA are generated in a similar manner, from longer RNA precursors. -Both are processed in the cytoplasm by an enzyme called Dicer. -Both form an RNP complex called RISC (RNA-Induced Silencing Complex). -Both are involved in a process called RNA-Induced Transcriptional Silencing (RITS) or RNA-Induced Gene Silencing.

What are the similarities between miRNA and siRNA?

inhibits translation of mRNA (cleave mRNA in animals)

What are the similarities or differences between miRNA and siRNA? miRNA action:

cleaves mRNA

What are the similarities or differences between miRNA and siRNA? siRNA action:

· Hyper-methylated guanosine residue that is linked to a 2'-O-pyrophosphate · 7-methyl guanosine · it is located at the 5' end of RNA

What is a cap?

7-methyl guanosine

What properties in this molecule indicate presence of a 5'-cap structure?

Plays a significant role in the interaction of mRNA with ribosomal RNA/its protein components during protein synthesis initiation. The cap is also involved in mRNA stability. If mRNA is less stable then there will be less transcription. It is also thought that capping is essential for mRNA synthesis- if this is true then transcription is further regulated by the availability of mRNAs.

What role does the cap play in regulating gene expression?

targets mRNA cleavage and degradation; represses translation (Essentially all aspects of biology, health and disease.)

What roles does miRNA play in gene regulation?

Protein synthesis · Uncapped/unmethylated mRNAs make poor templates · 7-methylguanosine facilitates binding of mRNAs to 40S ribosome · Chemical analogs of cap inhibit translation and ribosome binding of capped mRNA mRNA stability · Protects mRNA from degradation via nucleases · Stabilizes mRNA both in vivo and in cell-free extracts mRNA biosynthesis · Capping may be essential to mRNA synthesis · Processes may be coupled

Why does an mRNA need a cap?

Addition: Nucleotide phosphohydrolase removes a phosphate from the nascent RNA transcript. Guanylyltransferase then adds a Gppp group to the transcript and 2 phosphates are lost leaving a Gp. Methyltransferases I and II then methylate Gp to yield a 7mGP5' (7-methylguanosine) cap. removal: · Cap can be cleaved by tobacco nucleotide pyrophosphate · This cap removal is important for cloning and RACE techniques

look at video or description of the capping reaction process

Poly(A) tail · Poly(A) minus mRNAs decay much more rapidly than poly(A)-tailed mRNAs · Poly(A)-PABP complex protects mRNAs from rapid destruction in vitro 3'UTR · Histone mRNA 3' terminal stem loop · AU-rich elements (AUREs) o mRNA instability o Plays a critical role in HPV pathogenesis & oncogenesis o Following integration the AURE a the 3'-UTR of the viral mRNA is replaced with a host sequence that does not contain an AURE · Iron-responsive elements · Long-range stem loop of insulin-like growth factor II (IGF-II) mRNA coding region · C-myc · C-fos · Beta tubulin 5'-UTR and cap · C-myc o Longer 5'-UTR in chimeric c-myc-Ig is more stable than wild type c-myc mRNA · Adenovirus leader RNA o The leader stabilizes viral mRNAs approximately 5-fold at late times during viral infection · Caps o mRNAs without caps are less stable than capped mRNAs

mRNA STABILITY: Questions to be addressed: Which (cis-acting) sequence elements in mRNAs determine their stability?

Exoribonuclease · 3'-5' exo-RNase · A 33-kDa protein that degrades histone mRNA, protein-free Poly(A) but not poly(A) in a complex with PABP Endoribonucleases

mRNA STABILITY: Questions to be addressed: Which enzymes degrade mRNAs?

miRNA · Represses translation · Target mRNA cleavage and degradation siRNA · Cleave mRNA Similarities · Similar manner of generation · Processed by Dicer in cytoplasm · Form the RNP complex RISC · RNA-Induced Transcriptional Silencing or RNA-Induced Gene Silencing · siRNA = exogenous dsRNA that is taken up by cells · miRNA= ss & come from endogenous non-coding RNA · miRNA = complimentary sequence that induces mRNA cleavage instead of just repression of translation

microRNA and small interfering RNA: questions to be addressed: What roles does miRNA/siRNA play in gene regulation?

siRNA

short interfering RNA causing degradation of mRNA