Lesson 8 Flashcards
describe the central dogma in depth:
dna which can be replicated, is transcribed into RNA which is translated into proteins that are major players in regulating gene transcription
non-coding dna consists of what elements?
repetitive sequences (repetitive DNA and transposomes) and dna coding for noncoding RNA (long and small)
what are three types of small non-coding RNA?
miRNA, ribozymes, and tRNA
what does RNA interference lead to?
RNA silencing pathways
what is the overall effect when RNA silencing pathways are triggered?
negative regulation on gene expression
describe the origin of small interfering RNAs (siRNA):
endogenous or exogenous with respect to the cell they act on
what is the origin of miRNAs?
endogenous (cellular products) → any cells and somatic cells
what is the origin of Piwi-interacting RNAs?
endogenous → only germline cells
where was RNA interference first discovered?
C. elegans
the regulation of gene expression comes from an interaction within the genome at what level?
the level of DNA or chromatin modifications
what are most of the fundamental processes regulated by, such as development, differentiation, and cancer?
proteins together with microRNAs
why does the level of gene expression by RNA interference not occur at the level of initiation of transcription like with repressors?
mRNA is already transcribed but will not be expressed
are miRNAs endogenous or exogenous?
endogenous → small guide RNAs that repress the expression of target genes
how to miRNAs differ from siRNAs?
in biogenesis but not function
where are miRNAs abundant?
in ssRNA
describe the effect of miRNAs on mRNA:
one miRNA can regulate many different mRNAS
what does the presence of multiple binding sites for different miRNAs at the 3’ UTR determine?
that several different miRNAs can target the same 3’ region, meaning that each mRNA can be regulated by different miRNA
what two things does the complex regulation of miRNAs allow for?
for combinatorial control and system abundancy
how does the complex regulation of miRNAs allow combinatorial control?
more than one miRNA can regulate the same mRNA
how does the complex regulation of miRNAs allow system abundance?
losing one miRNA due to mutation or deletion does not impact the regulation of the genes as there are many other that can get the job done
what is the structure of miRNAs?
21-24 nt regulatory RNAs with a hairpin secondary structure
list some functions of miRNA:
participate in the regulation of many important processes such as the regulation of signaling pathways, apoptosis, metabolism, development, cancer → they are important as well as proteins for the regulation of gene transcription at the post-transcriptional level
since an miRNAs mechanism of action is based on matching, what can occur?
the targets in the genome can be predicted by sequencing them
what are two roles of miRNA in biological processes?
help to confer robustness to transcriptional programs operated by regulatory proteins and since they do not interfere with the product, their role can also be to regulate aberrant transcripts in the cell
at what point during development does miRNA expression increase?
during embryonic development
in animals, what correlation has been found with miRNA?
the more complex the organism, the more diverse the miRNA populaiton
genes with tissue-specific expression have a longer 3’ UTR meaning what?
they have more miRNA binding sites → the genes encoding for proteins specifically expressed in a tissue during a certain step of differentiation require more regulation by miRNA
LEARN RNAi PATHWAY
what are miRNA’s function essential for?
sustaining life
what is the prototype of genes coding for miRNA in C. elegans?
lin-4
what does Lin-4 miRNA regulate?
lin-14 mRNA
why are there multiple binding sites in the 3’ UTR?
we have different levels of complimentary, so no perfect match is needed (but some regions need to be matched)
describe the structure of Lin-4 miRNA:
transcribed as a single molecule and thanks to its intra-strand complimentary regions formed a double-stranded RNA (forms kind of a hairpin structure)
where are the genes encoding miRNAs located?
the nucleus
what is the final product of transcription?
pri-miRNA
what is pri-miRNA?
a single stranded molecule that has a 5’ end and a 3’ end, and a quite extended region of intrastrand homology with a small loop a the end
in the nucleus, what is pri-miRNA processed by?
Drosha
in mammals, what is Drosha coupled with?
DGCR8
what is the function of Drosha + DGCR8? (after transcription)
responsible for the first major processing, which consists of cleavage and generation of the 3’ overhang
after pri-miRNA is processed by Drosha, what does it become?
pre-miRNA
where must the regulation of miRNAs occur?
cytoplasm
how do pre-miRNAs get to the cytoplasm?
exported from the nucleus through nuclear pores by RAN-GTP/exportin 5 complex
what enzyme processes pre-miRNA in the cytoplasm?
DICER
what is the function of DICER?
the enzyme which is responsible for trimming the pre-miRNA
what is the main modification from pre-miRNA to mature miRNA?
the loop is lost and the 3’ overhang on both sides of the strands are formed
which are the only types of mature miRNA included in RISC complexes?
double strand structure of mature miRNA
once miRNA is mature, describe the structure:
two different strands called Duplex miRNA/miRNA*→ one will be retained by the RISC complex and the other will be released and degraded
in what three ways are miRNAs present in the genome?
in an intergenic position, intragenic position, or in clusters
describe in intergenic position:
transcription is regulated by specific elements such as promotors (is a single transcription unit)
describe an intragenic position:
transcription miRNA is regulated by the “host” gene elements → miRNA are included in introns so transcription will occur under the control of the promotor of the gene in which they are contained
describe clusters in relation to miRNA genes:
in Drosophila: processed as polycistroninc RNAs and can be transcribed as polycistronic mRNA
in the intragenic position, are the miRNAs going to regulate the transcript in which they are included?
yes but not always
describe Drosha:
a nuclear enzyme with RNAse activity (trims long transcripts)
in drosophila, what does Drosha work with instead of DGCR8?
Pasha
describe the processing of pri-miRNA:
during processing all the inferior stem is lost while all the superior stem is retained, the loop is lost as well
describe the structure of Drosha:
double-stranded RNA binding domains, two domains with RNAse activity, a N-terminal domain and a nuclear localization signal
why does drosha need a partner (DGCR8 or Pasha)?
it is not able to selectively cut the pri-miRNA the correct way
where is the 3’ overhang on pre-miRNA?
2 nucleotide overhang on the 3’ end
where does dicer cut the miRNA?
recognizes the double strand end of the loop and cuts both ends of the duplex
what does RISC stand for?
RNA induced silencing complex
describe the structure of dicer:
helices domain, two RNAse III domains, a dsRNA binding domain and a PAZ domain
when is the RISC complex transformed into an active form?
only when there is the unwinding of the siRNA or miRNA duplex and one strand is lost → when the miRNA is still bound the complex is inactive
what is the mechanism of action in RISC assembly?
RISC recognizes the target mRNA and this recognition is mediated by the match between miRNA and the mRNA
name two ways in which miRNA silencing can occur:
if the cut of mRNA is in a very specific position (mRNA is cut at the level of nucleotides complimentary to th exposition of nucleotide 10 and 11 in the miRNA) OR the inhibition of translation due to the absence of a perfect match
what is the choice between the two silencing mechanisms of miRNA dependent on?
the level of complementarity between miRNA / siRNA and the target mRNA
name 5 possible mechanisms for miRISC-mediated repression (inhibition of translation):
compete for cap binding, promote degradation, compete for eiF2/60S, block circulation, or cause ribosome dropoff
