Module 7 Flashcards
How is miRNA synthesized?
- precursor primary miRNA (pri-mRNA) is encoded by the genome (synthesized by RNA polymerase II) -
- in the nucleus, primRNA is cut by Drosha into single stem loop structure (pre-mRNA)
- in the cytosol, dicer forms a 19-25 nucleotide mRNA: mRNA duplex with NO stem loop structure (miRNA)
What protein does RISC contain?
Argonaute protein
What are the two forms of RISC? What does RISC stand for?
RNA induced silencing complex
two forms: SiRNA and MiRNA
Describe SiRNA ?
- passenger strain leaves
- siRISC: guide strand
- guide strand is a perfect match for the target RNA = cleavage!
Describe miRNA
- passenger strand is discarded, miRISC: guide strand
- incomplete complemntary binding of guide strand to target miRNA
= close enough match: this can lead to mRNA degradation, mRNA repression, or cleavage
How is siRISC synthesized?
- double stranded RNA cleaved by dicer ao produce siRNAs
- combines with proteins to form RISC which pairs with and cleaves mRNA
What is transcriptional gene silencing?
- RNAi condenses chromatin to suppress transcription, mRNA is not made
or:
other siRNAs bind to complementary sequences in DNA and attract methylating enzymes which methylate DNA or histones and inhibit transcription (uses RITS: in the nucleus)
What is the difference between RISC and RITS?
RITS: RNA-induced initiation of transcriptional silencing - In the nucleus, used in siRNA methylation for transcriptional silencing
RISC: in the cytoplasm: used for post transcriptional silencing
How does RNAi (RNA interference) direct localized repressive chromatin formation?
- siRNA duplexes are loaded into a nuclear form of RISC called RITS
- RITS are effector complexes targted to homologous sequences by base pairing interactions involving guide strand of siRNA
RITS ,edits gene silencing. via heterochromatin formation
What is RITS job?
- mediates gene silencing via heterochromatin formation (heterochromatin is highly packed, cannot be transcribed)
What are the two forms of chromatin?
- heterochromatin: the densely packed, inactive form of chromatin
- euchromatin: the open active form of DNA
What are some methods of histone modification?
- phosphorylation, methylation, acetylation
What does collinearity mean? Is it accurate?
- the number of nucleotides in the gene is proportional to the number of amino acids in the protein
- more accurate for prokaryotes than eukaryotes
What is the structure of the mRNA in prokaryotes?
- 5’ untranslated region, shine-dalgarno sequence (prokaryotes only) - start codon - protein coding region - stop codon - 3’ untranslated region
Describe the structure of the premRNA and mRNA in eukaryotes
pre-mRNA: 5’ cap - start codon - protein coding region (with exons and introns) - stop codon - 3’UTR and poly A tail
- mRNA: 5’ cap - 5’UTR - start codon - protein coding region - stop codon - 3’UTR - Poly A tail
What is the major type of intron?
- nucelar pre-mRNA
- protein encodingg genes In the nucleus of eukaryotes: spliceosomal splicing mechanism
Describe the gene organization In prokaryotes
prokaryotic protein doing genes usually found in a continuous array in DNA called an operon
- a single transcription start site for multiple genes
Describe the gene organization in eukaryotes
- each gene is transcribed from its own start site yielding a pre-mRNA which is processed into an mRNA that yields a single protein
Do prokaryotes generally have many introns?
- introns are generally rare : many mRNA strands are synthesized into proteins as they are being transcribed
How is pre-mRNA processed in eukaryotes after transcription?
1) capping at 5’ end
2) polyadenylation of the 3’ end (poly A tail)
3) introns spliced out
What do the post transcriptional modifications in eukaryotes do?
- 5’ cap: necessaryy for initiation of translation provides stability (protects mRNA from degradatION) , enhances RNA splicing, transport of mRNA from nucleus,
- poly A tail : increases mRNA stability, aid in export of mRNA from nucleus, facilitates binding of ribosome to mRNA
- RNa splicing: removes introns, multiple proteins can be produced, facilitates export of mRNA into cytoplasm
What is 5’ capping?
- a methylated guanine is added to the 5’ end of pre-mRNA
- necessary to initiate translation, protects mRNA from degradation, enhances RNA splicing, helps in exporting mRNA from the nucleus
What is 3’ polyadenylation?
- pre-mRNA is cleaved 11-30 nucleotides downstream of consensus sequence in the 3’ UTR region
- ~50-250 Adenine nucleotides are added
- in premRNA a poly A tail is added by cleavage and polyadenylaion
- necessary for efficient translation, protects mRNA from degradation
NOTE: this also terminates transcription
Which consensus sequences does splicing require? Why are they important?
- 5’ splice site, 3’ splice site, branch point
- consensus sequences used by spliceosome to recognize/remove introns
What process occurs with the spliceosome in eukaryote pre-mRNA processing?
- splicing involves brwaking and reforming bonds
-introns are removed in the forms of lariats, and the two exons are joined together by two successive reactions (transesterification)
Where does splicing take place?
- splicing occurs on the spliceosome: a ribonucleoprotein with 300 proteins and 5 snRNAs
- has 5 snRNPS (snRNA+protien) = U1, U2, U4, U5, U6
- U1 and U2 binds to the 5’ splice site and the branching point to form a lariat and exons, lariat is removed and the exons are joined by transesterification reactions
What does it mean for RNA polymerase II to have functional coupling of transcription and processing?
- coupling of mRNA transcription and processing by RNA polymerase II
- coupling of events are mediated by the tail or C-terminal repeat domain (CTD) of the largest subunit of polymerase II
- mRNA processing enzymes are recruited to the CTD of Pol II during transcription
When is the 5’CAP added to the pre-mRNA? When do the capping enzymes appear to do this?
- as soon as the 5’ end emerges from the polymerase (first to come out!)
- capping enzymes are recruited to the CTD during early stages of transcription
How does the pre-mRNA get directed to splicing after being capped?
- Pol II mediates functional coupling of transcription to splicing by directing the basement pre-mRNA into spleceosome assembly
At what stage is the spliceosome assembled?
- occurs co transcriptionally while RNA pol II is still transcribing the template
- components of the spliceosome are recruited while the transcription of pre-mRNA Is occurring
- the CTD of pol II interacts directly with splicing proteins to recruit them to RNA
Considering that transcription is terminated when polyadenylation occurs, describe why this process occurs
- polyadenylation factors are recruited to the CTD of Pol II
- RNA is cleaved t the poly A 3’ cleavage site
- degradation of the remaining RNA by Rat 1 terminates transcription
What processes are coordinated by CTD?
The CTD of RNA Pol II coordinates all 3 processes of mRNA processing (capping, polyadenylation, and splicing)
What is alternative processing?
- Human genome sequence: ~20,000 protein encoding genes
- proteonome is more complicated because one gene can give rise to thousands of proteins
what are the ways that pre-mRNA can be processed (alternate processing)?
- alternative splicing; mRNA can be spliced in many different ways
- alternative poly A site: poly A tail can be added at 3 different 3’ cleavage sites: each cleavage site produces different sizes of similar proteins
What are different proteins formed from the same gene exons called?
isoforms of proteins
what are the four alternative splicing patterns?
- exon skipped
- intron retained
- alternative 3’ or 5’ splice site
- mutual exclusive exon
what does regulation of alternative processing refer to?
- proteins could be functional or non functional depending on which part of the exon is included
What is the role of genetic diseases in splicing?
- many genetic diseases arise from mutations that affect pre-mRNA splicing
what are some consequences of mutations impacting splice sites?
- may disrupt consensus sequences so that 5’ or 3’ splice site is unrecognized
- may produce new 3’ or 5’ splice sites
- may initiate use of existing cryptic 5’ or 3’ splice site
- may also lead to exon skipping or intron retention (complete or partial) which can result in non functional proteins or create mRNA instability (leading to protein degradation)
What is considered central to the functioning of the spliceosome?
- snRNPS
describe beta thalassemia
- caused by mutations in the B-globin (involved with hemoglobin) gene many of which lead to incorrect splicing
- one of the most common genetic diseases: leads to increased blood cell destruction leading to anemia
Describe RNA editing
- occasionally a gene Is found that does not match the nuceltodie sequence of its RNA product
- editing alters the coding information of mRNA transcripts
ways to do this: substitution editing or insertion editing
what is substitution editing?
- chemical alteration of individual nucleotides by specific enzymes
What is insertion editing?
- addition of U nucleotides occurs via: cleavage of the mRNA, adding the U nucleotides, and fixing the ends through ligase
- this Is guided by guideRNA which base pair as best they can with the mRNA to be edited and serve as a template for addition of nucleotides
