Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

zBCHM > Alison series > Flashcards

Alison series Flashcards

1
Q

splicing

A

RNA is modified in the nucleus by additions to the 5’ and 3’ ends and by splicing to remove the introns. The splicing event requires breakage of the exon-intron junctions and joining of the ends of the exons. Mature mRNA is transported through nuclear pores to the cytoplasm, where it is translated.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what is pre-mRNA

A

Pre-mRNA is used to describe the nuclear transcript that is processed by modification and splicing to give an mRNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is RNA splicing

A

RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what is hnRNA

A

Heterogeneous nuclear RNA (hnRNA) comprises transcripts of nuclear genes made by RNA polymerase II; it has a wide size distribution and low stability.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is an hnRNP

A

An hnRNP is the ribonucleoprotein form of hnRNA (heterogeneous nuclear RNA), in which the hnRNA is complexed with proteins. Since pre-mRNAs are not exported until processing is complete, hnRNPs are found only in the nucleus.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

The 5′ End of Eukaryotic mRNA Is Capped

A

•
A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link during transcription.
•
The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation
•
The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Nuclear splice junctions are short sequences

A

•
Splice sites are the sequences immediately surrounding the exon-intron boundaries.
•
The 5´ splice site at the 5´ (left) end of the intron includes the consensus sequence GU.
•
The 3´ splice site at the 3´ (right) end of the intron includes the consensus sequence AG.
•
This is called the GU-AG rule
The ends of nuclear introns are defined by the GU-AG rule.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Nuclear splice junctions are short sequences

A

•
Splice sites are the sequences immediately surrounding the exon-intron boundaries. They are named for their positions relative to the intron.
•
The GU-AG rule (originally called the GT-AG rule in terms of DNA sequence) describes the requirement for these constant dinucleotides at the first two and last two positions of introns in pre-mRNAs.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Splice junctions are read in pairs

A

•
Splicing depends only on recognition of pairs of splice junctions.
•
All 5´ splice sites are functionally equivalent, and all 3´ splice sites are functionally equivalent.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Correct splicing removes three introns by pairwise recognition of the junctions

A

y

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Alternative splicing contributes to structural and functional diversity of gene products

A

y

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Pre-mRNA Splicing Proceeds through a Lariat

A

•
Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site.
•
The branch sequence is conserved in yeast but less well conserved in multicellular eukaryotes.
•
A lariat (an RNA intermediate with a circular structure) is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron.
•
A transesterification reaction breaks and makes chemical bonds in a coordinated transfer so that no energy is required.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what is a lariat

A

A lariat (an RNA intermediate with a circular structure) is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

pre-mRNA splicing proceeds through a lariat

A

•
A lariat is formed when the intron is cleaved at the 5´ splice site, and the 5´ end is joined to a 2´ position at an A at the branch site in the intron.
•
The intron is released as a lariat when it is cleaved at the 3´ splice site, and the left and right exons are then ligated together.
•
The 5´ and 3´ splice sites and the branch site are necessary and sufficient for splicing.
•
The branch sequence is conserved in yeast but less well conserved in higher eukaryotes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what is snRNA

A

•
A small nuclear RNA (snRNA) is one of many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

snRNAs are required for splicing

A

•
A small nuclear RNA (snRNA) is one of many small RNA species confined to the nucleus; several of the snRNAs are involved in splicing or other RNA processing reactions.
•
snRNPs (snurps) are small nuclear ribonucleoproteins (snRNAs associated with proteins)
•
The spliceosome is a complex formed by the snRNPs that are required for splicing together with additional protein factors.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

what is snRNPs

A

•

snRNPs (snurps) are small nuclear ribonucleoproteins (snRNAs associated with proteins)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what is spliceosome

A

The spliceosome is a complex formed by the snRNPs that are required for splicing together with additional protein factors.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what 5 snRNPs involved in splicing

A

•
The five snRNPs involved in splicing are U1, U2, U4, U5, and U6. These make up almost half the mass.
•
Together with some additional proteins, the snRNPs form the spliceosome.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

U1 snRNA

A

U1 snRNA has a base-paired structure that creates several domains.
The 5’ end remains single stranded and can base pair with the 5’ splicing site.
U1 snRNA selects the donor splicing junction Mutations that abolish function of the 5’ splicing site can be suppressed by compensating mutations in U1 snRNA that restore base pairing

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Commitment of Pre-mRNA to the Splicing Pathway – U1 snRNP initiated

A

•
U1 snRNP initiates splicing by binding to the 5´ splice site by means of an RNA-RNA pairing reaction
•
The E complex (commitment complex) contains U1 snRNP bound at the 5´ splice site, the protein U2AF bound to a pyrimidine tract between the branch site and the 3´ splice site, and SR proteins connecting U1 snRNP to U2AF

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Commitment of Pre-mRNA to the Splicing Pathway

A

•
In multicellular eukaryotic cells, SR proteins play an essential role in initiating the formation of the commitment complex.
•
An SR protein has a variable length of n Arg-Ser-rich region

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

The Spliceosome Assembly Pathway

A

•
The commitment complex (U1 and U2) progresses to pre-spliceosome (the A complex) in the presence of ATP.
•
Recruitment of U5 and U4/U6 snRNPs converts the pre-spliceosome to the mature spliceosome (the B1 complex).
•
The B1 complex is next converted to the B2 complex in which U1 snRNP is released to allow U6 snRNA to interact with the 5′ splice site.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

5 snRNPs form the spliceosome

A

•
Binding of U5 and U4/U6 snRNPs converts the A complex to the B1 spliceosome, which contains all the components necessary for splicing.
•
The spliceosome passes through a series of further complexes as splicing proceeds.
•
Release of U1 snRNP allows U6 snRNA to interact with the 5´ splice site, and converts the B1 spliceosome to the B2 spliceosome.
•
When U4 dissociates from U6 snRNP, U6 snRNA can pair with U2 snRNA to form the catalytic active site.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
U6 snRNA can pair with either U4 or U2
U6-U4 pairing is incompatible with U6-U2 pairing. When U6 joins the spliceosome it is paired with U4. Release of U4 allows a conformational change in U6; one part of the released sequence forms a hairpin, and the other part pairs with U2.
26
snRNA pairing is important in splicing
Splicing utilises a series of base-pairing reactions between snRNAs and splice sites.
27
E complex
U1 (5’ splice site) + U2AF (Py tract and 3’ splice site)
28
A complex
+ ATP to U2 (branch site)
29
B1 complex
U4/U6 + U5
30
B2 complex
U1 leaves, then U4. U2 binds to U6 (5’ splice site)
31
C1
Transesterification 1
32
C2
Transesterification 2
33
3′ UTR (3′ untranslated region)
The untranslated sequence downstream from the coding region of an mRNA
34
5′ UTR (5′ untranslated region)
The untranslated sequence upstream from the coding region of an mRNA.
35
stem-loop
A secondary structure that appears in RNAs consisting of a base-paired region (stem) and a terminal loop of single-stranded RNA. –Both are variable in size.
36
Messenger RNAs Are Unstable Molecules
* mRNA instability is due to the action of ribonucleases. * Ribonucleases differ in their substrate preference and mode of attack. * endoribonuclease – A ribonuclease that cleaves an RNA at an internal site(s). * exoribonuclease – A ribonuclease that removes terminal ribonucleotides from RNA.
37
endoribonuclease
A ribonuclease that cleaves an RNA at an internal site(s).
38
•exoribonuclease
– A ribonuclease that removes terminal ribonucleotides from RNA.
39
Messenger RNAs Are Unstable Molecules
* mRNA decay – mRNA degradation, assuming that the degradation process is stochastic. * Differential mRNA stability is an important contributor to mRNA abundance and therefore the spectrum of proteins made in a cell. * steady state (molecular concentration) – The concentration of population of molecules when the rates of synthesis and degradation are constant.
40
•mRNA decay
– mRNA degradation, assuming that the degradation process is stochastic.
41
•steady state (molecular concentration)
– The concentration of population of molecules when the rates of synthesis and degradation are constant.
42
Eukaryotic mRNAs Exist in the Form of mRNPs from Their Birth to Their Death
* mRNA associates with a changing population of proteins during its nuclear maturation and cytoplasmic life. * Some nuclear-acquired mRNP proteins have roles in the cytoplasm. * A very large number of RNA-binding proteins (RBPs) exist, most of which remain uncharacterized. * Different mRNAs are associated with distinct, but overlapping, sets of regulatory proteins, creating RNA regulons.
43
Most Eukaryotic mRNA is Degraded via Two Deadenylation-Dependent Pathways
* The modifications at both ends of mRNA protect it against degradation by exonucleases. * poly(A) binding protein (PABP) – The protein that binds to the 3′ stretch of poly(A) on a eukaryotic mRNA. * The two major mRNA decay pathways are initiated by deadenylation catalyzed by poly(A) nucleases. * Deadenylation may be followed either by decapping and 5′ to 3′ exonuclease digestion, or by 3′ to 5′ exonuclease digestion. * The decapping enzyme competes with the translation initiation complex for 5′ cap binding. * cytoplasmic cap-binding protein – A component of the eukaryotic initiation factor 4F (eIF4F) that binds the 7-methyl guanosine cap at the 5′ end of eukaryotic mRNA. * The exosome, which catalyzes 3′ to 5′ mRNA digestion, is a large, evolutionarily conserved complex.
44
•poly(A) binding protein (PABP) –
The protein that binds to the 3′ stretch of poly(A) on a eukaryotic mRNA.
45
decapping enzyme
•The decapping enzyme competes with the translation initiation complex for 5′ cap binding.
46
•cytoplasmic cap-binding protein –
A component of the eukaryotic initiation factor 4F (eIF4F) that binds the 7-methyl guanosine cap at the 5′ end of eukaryotic mRNA.
47
mRNA-Specific Half-Lives Are Controlled by Sequences or Structures within the mRNA
* Specific cis-elements in an mRNA affect its rate of degradation. * Destabilizing elements (DEs) can accelerate mRNA decay, while stabilizing elements (SEs) can reduce it. * mRNA degradation rates can be altered in response to a variety of signals. * iron-response element (IRE) – A cis sequence found in certain mRNAs whose stability or translation is regulated by cellular iron concentration.
48
•iron-response element (IRE
) – A cis sequence found in certain mRNAs whose stability or translation is regulated by cellular iron concentration.
49
A Small Aside: Quality Control of mRNA Translation
* Nonsense-mediated decay (NMD) targets mRNAs with premature stop codons. * Targeting of NMD substrates requires a conserved set of Upf and SMG proteins. * Recognition of a termination codon as premature involves unusual 3′ UTR structure or length in many organisms and the presence of downstream exon junction complexes (EJC) in mammals. * Nonstop decay (NSD) targets mRNAs lacking an in-frame termination codon and requires a conserved set of SKI proteins. * No-go decay (NGD) targets mRNAs with stalled ribosomes in their coding regions.
50
•Nonstop decay (NSD)
targets mRNAs lacking an in-frame termination codon and requires a conserved set of SKI proteins.
51
•No-go decay (NGD)
targets mRNAs with stalled ribosomes in their coding regions.
52
Some Eukaryotic mRNAs Are Localized to Specific Regions of a Cell
* Localization of mRNAs serves diverse functions in single cells and developing embryos. * mRNP granules – Large mRNA-containing cytoplasmic particles, such as processing bodies (P bodies), stress granules, and neuronal granules.
53
•mRNP granules –
Large mRNA-containing cytoplasmic particles, such as processing bodies (P bodies), stress granules, and neuronal granules.
54
•Degradation may occur within discrete cytoplasmic particles called processing bodies (PBs).
•A variety of particles containing translationally repressed mRNAs exist in different cell types. –maternal mRNA granules – Oocyte particles containing translationally repressed mRNAs awaiting activation later in development. –neuronal granules – Particles containing translationally repressed mRNAs in transit to final cell destinations. –stress granules – Cytoplasmic particles, containing translationally inactive mRNAs, that form in response to a general inhibition of translation initiation.
55
•A variety of particles containing translationally repressed mRNAs exist in different cell types.–maternal mRNA granules –
Oocyte particles containing translationally repressed mRNAs awaiting activation later in development.
56
•A variety of particles containing translationally repressed mRNAs exist in different cell types––neuronal granules –
Particles containing translationally repressed mRNAs in transit to final cell destinations.
57
•A variety of particles containing translationally repressed mRNAs exist in different cell types–stress granules –
–stress granules – Cytoplasmic particles, containing translationally inactive mRNAs, that form in response to a general inhibition of translation initiation.
58
3 main mechasnisms of mRNA localization
1. pattern formation and fate specialization in oocytes and embryos 2. generalization of different daughter cells in assymetric cell division 3. compartmentalization of a cell into specialized regions.
59
Some Eukaryotic mRNAs Are Localized to Specific Regions of a Cell
* Localization requires cis-elements on the target mRNA and trans-factors to mediate the localization. * zipcode (or localization signal) – Any of the number of mRNA cis elements involved in directing cellular localization. * The predominant active transport mechanism involves the directed movement of mRNPs along cytoskeletal tracks.
60
•zipcode (or localization signal) –
Any of the number of mRNA cis elements involved in directing cellular localization.
61
RNA function
``` RNA functions as a regulator by forming a region of secondary structure (either inter- or intramolecular) that changes the properties of a target sequence ```
62
Noncoding RNAs Can Be Used to Regulate | Gene Expression
• Vast tracts of the eukaryotic genome are transcribed. • antisense gene – A gene that codes for an (antisense) RNA that has a complementary sequence to an RNA that is its target. • antisense RNA – RNA that has a complementary sequence to an RNA that is its target.
63
transcriptional interference (TI)
• A regulator RNA can function by forming a duplex region with a target RNA. • The duplex may block initiation of translation, cause termination of transcription, or create a target for an endonuclease. • Transcriptional interference (TI) occurs when an overlapping transcript on the same or opposite strand prevents transcription of another gene.
64
Bacteria Contain Regulator RNAs
• Bacterial regulator RNAs are called sRNAs. • Tandem repeats can be transcribed into powerful antiviral RNAs. • CRISPR – Clusters of regularly interspersed short palindromic repeats in bacteria and Archea that are transcribed and processed into short RNAs that function in RNA interference
65
``` Mechanism of the CRISPR pathway • Effector complex targets invader DNA for cleavage ```
y
66
Small RNAs Are Widespread Regulators in | Eukaryotes
• Eukaryotic genomes code for many short (~22 base) RNA molecules. • RNA interference (RNAi) – A process by which short (21 to 23) nucleotide antisense RNAs, derived from longer double-stranded RNAs, can modulate expression of mRNA by translation inhibition or degradation.
67
• RNA interference (RNAi) –
• RNA interference (RNAi) – A process by which short (21 to 23) nucleotide antisense RNAs, derived from longer double-stranded RNAs, can modulate expression of mRNA by translation inhibition or degradation.
68
``` All small RNAs are bound by an Argonaute protein ```
``` (a) Ago proteins are characterized by PIWI and PAZ domains. A so-called MID domain is located between the PAZ and the PIWI domain. (b) Crystal structure of the Argonaute protein of Thermus thermophilus. Argonaute proteins are bi-lobal proteins. The MID domain (green) anchors the 5′ end and the PAZ domain (blue) the 3′ end of the small RNA. The PIWI domain (orange) is structurally similar to RNase H and some PIWI domains can cleave target RNAs endonucleolytically ```
69
MicroRNA Biogenesis Drosha and dicer
• Drosha – An endonuclease that processes doublestranded primary RNAs into short, ~70 base pair precursors for Dicer processing. • Dicer – An endonuclease that processes doublestranded precursor RNA to 21 to 23 nucleotide RNAi molecules.
70
dicer
• Dicer – An endonuclease that processes doublestranded precursor RNA to 21 to 23 nucleotide RNAi molecules.
71
drosha
• Drosha – An endonuclease that processes doublestranded primary RNAs into short, ~70 base pair precursors for Dicer processing.
72
Dicer
``` (a) Dicer is characterized by a number of domains namely a DEAD box helicase domain, a domain of unknown func(on (DUF283), a PAZ domain, two RNase III domains and a double stranded RNA-­‐binding domain. (b) Structure of Dicer from Giardia intes(nalis. Dicer binds the end of the long double stranded RNA (shown in yellow) and cleaves about 21 nucleo(des upstream resul(ng of a 21-­‐nucleo(de double stranded RNA product. ```
73
miRNA biogenesis
``` Genes encoding miRNAs are initially transcribed by RNA polymerase II or III to generate the pri-miRNA transcripts within the nucleus. The stem-loop structure of the pri-miRNA is recognized and cleaved on both strands by a microprocessor complex, which consists of the nuclear RNase III enzyme Drosha and an RNAbinding protein, DGCR8, to yield a pre-miRNA 60–70 nt in length. The pre-miRNA is then exported from the nucleus through a nuclear pore by exportin-5 in a Ran-GTPdependent manner and processed in the cytoplasm by the RNase III Dicer– TRBP. Sliced RNA strands are further unwound by an RNA helicase. One strand of the miRNA/ miRNA* is then preferentially incorporated into the miRNP and will guide the miRNP to a target mRNA in a sequencespecific manner. ```
74
How Do microRNAs Work?
``` • MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs. • Depending on the degree of complementarity between the miRNA and the target RNA, different pathways can be employed High complementarity between the two RNAs = endoribonucleolytic cleavage. This is frequently found in plants. Partial complementarity (typical in animals) leads to either translational repression or mRNA degradation. ```
75
How were miRNAs discovered?
``` • The first miRNA (lin-­‐4) was discovered in C. elegans in 1993 • The second (let-­‐7) was found in 2000, and shortly aXerwards many miRNAs in many species were discovered. ```
76
``` What do most miRNAs do? ```
``` • miRNAs have roles in many, if not all, cellular pathways • Most miRNAs occur in families and dele(on of only one miRNA will have li\le or no effect • Some(mes even dele(on of the en(re family has no obvious effect • It can be very difficult to determine the target gene(s) – There are many different predic(on algorithms but none are ideal and most give many apparent false posi(ves ```
77
Where does the long dsRNA come from?
``` • Exogenously supplied • Viruses • Endogenous loci • Transgenes ```
78
RNAi – exogenously supplied dsRNA
``` • Discovered in C. elegans by Andy Fire and Craig Mello in the 1990s and earned them the Nobel prize • Dicer cleavage • siRNA duplex unwound • Argonaute/RISC incorporation • Target cleavage and then degradation (Xrn1 and the exosome) ```
79
RNAi – exogenously supplied dsRNA
``` • Can be used in almost all organisms • Used for: – Making knock downs of genes (gene function analysis) – Therapeutics • Wet age-related macular degeneration • Diabetic macular oedema • Hep B • AIDS • Tumours ```
80
Anti-viral siRNAs
• Many viruses go through a dsRNA intermediate at many points during their replication • These dsRNAs can act as Dicer targets and trigger the RNAi pathway • Occurs in Drosophila, plants, C. elegans (and maybe mammals)
81
FHV example
Following entry and uncoating of flock house virus (FHV) virions, the genomic positive-strand RNA ((+)RNA) serves as both mRNA for the translation of viral RNA-dependent RNA polymerase (RdRP) and as a template for the synthesis of antigenomic negative-strand RNA ((−)RNA). The resulting double-stranded RNA (dsRNA) formed between the 5′-terminal nascent progeny (+)RNA and the (−)RNA template is recognized by Dicer 2 (DCR2) and cleaved into small interfering RNAs (siRNAs). The viral siRNAs are assembled with Argonaute 2 (AGO2) into the RNA-induced silencing complex (RISC) and used to guide specific clearance of FHV RNAs. As a counter-defence, FHV encodes a viral suppressor of RNA silencing, the B2 protein, which targets two steps in this immune pathway: inhibition of viral siRNA production by binding to viral RdRP and the viral dsRNA precursor, and sequestration of viral siRNAs by binding duplex siRNAs.
82
Endo siRNAs
``` • siRNAs that are generated from endogenous loci • Found in all organisms, but their function remains largely unknown – Developmental plasticity – Learning and memory – Heterochromatin formation ```
83
Heterochromatin Formation Requires siRNAs
• siRNAs can promote heterochromatin formation. • RNA-dependent RNA polymerase (RDRP) –A component of the RITS complex that copies the heterochromatin ncRNA that is then used to silence heterochromatin transcription. • RITS (RNA-induced transcriptional silencing) – A complex that uses short single-stranded siRNA to repress heterochromatin transcription.
84
siRNAs and their role in transgenerational silencing
• Epigenetic marks are mostly erased between generations – this is to restore pluripotency to the cells of the developing embryo • Sometimes the marks are not fully erased: siRNAs are transported to the nucleus where they interact with the chromatin to establish repressive histone marks (we think!)
85
C. elegans as a tool to study transgenerational silencing
• If we count the number of GFP silenced worms each generation we can get an accurate representation of the transgenerational silencing that is occurring • We can test various genetic mutants to see what effect they have on the inheritance of the silencing signal
86
piRNAs
``` • Piwi-associated RNAs • Proteins but not RNAs conserved across a broad range of species • Major role in transposon defence • Role in (male) fertility ```
87
piRNA biogenesis in C. elegans
``` • No ping pong amplification • RNA-dependent RNA polymerase (RdRP) amplification Not all piRNAs are transposon derived ```
88
Summary | L14
``` • RNA plays a vital role in the cell – Information transfer: DNA-proteins – Splicing influences protein form and function – RNA molecules can be regulatory – RNA can provide resistance against cellular attack (external and internal) – RNA can affect the packaging of DNA ```

zBCHM (5 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions