Download Genetic regulation in eukaryotes

RNAs
Ribonucleic acids (RNAs)
INTRODUCTION
SLIDEs 1+2 Ancient RNA World Proteins cannot self-replicate, and many evolutionary geneticists
consider that autocatalytic nucleic acids must have pre-dated proteins and were able to replicate without the help
of proteins. The RNA World Hypothesis developed from ideas proposed by Alexander Rich and Carl Woese in
the 1960’s. It imagines that RNA had a dual role in the earliest stages of life, acting as both the genetic material
(with the capacity for self-replication) and also as effector molecules, such as proteins today. Both roles are still
evident today: some viruses have RNA genomes, and non-coding RNA molecules can work as effector
molecules with catalytic activity (ribozymes). Another observation consistent with RNAs being the first nucleic
acid is that deoxyribonucleotides are synthesized from ribonucleotides in cellular pathways (by reverse
transcription). As well as storing genetic information, RNA has been imagined to have been used subsequently
to synthesize proteins from amino acids. RNA has a rather rigid backbone and so is not very well suited as an
effector molecule. Proteins are much more flexible and also offer more functional variety because the 20 amino
acids can have widely different structures and offer more possible sequence combinations. The replacement of
RNA with DNA as an information storage molecule provided significant advantages. DNA is much more stable
than RNA, and so better suited for this task. The sugar residues of the DNA lack the 2’OH group on ribose
sugars that makes RNA prone to hydrolytic cleavage. Greater efficiency could be achieved by separating the
storage and transmission of genetic information (DNA) from protein synthesis (RNA). All that was needed was
the development of a reverse transcriptase so that DNA could be synthesized from deoxyribonucleotides by
using an RNA template.
TYPES of RNAs
RNAs for PROTEIN SYNTHESIS SLIDES 3-6
SLIDE 4 (1) Messenger RNAs have already discussed in detail in the lecture on Genetic
Regulation in Eukaryotes. Briefly:
Prokaryotes:
• transcription and translation is coupled in space and time
• instability: half life time 1-3 min
• polycistronic mRNAs (operons: more than one genes on a single mRNA molecule).
Eukaryotes
• 1 gene encodes 1 protein
• pre-mRNAs are processed: splicing, capping, polyA tail
SLIDE 5 (2) Ribosomal RNA genes In addition to the two mitochondrial rRNA molecules
(12S and 16S rRNA), there are four types of cytoplasmic rRNA, three associated with the
large ribosome subunit (28S, 5.8S, and 5S rRNAs) and one with the small ribosome subunit
(18S rRNA). The 5S RNA genes occur in small gene clusters, the largest being a cluster of 16
genes on chromosome 1q42, close to the telomere. Only a few 5S RNA genes are functional,
and there are many dispersed pseudogenes. The 28S, 5.8S, and 18S rRNAs are encoded by a
single multigenic transcription unit that is tandemly repeated to form megabase-size
ribosomal DNA arrays (about 30-40 tandem repeats, or roughly 100 rRNA genes) on the short
arm of the acrocentric human chromosomes 13, 14, 15, 21, and 22.
SLIDE 6 (3) Transfer RNA genes The 22 mitochondrial tRNAs are made by 22 tRNA genes
in mtDNA. 516 human tRNA genes are known that make cytoplasmic tRNA with defined
anticodon specificity. The genes can be classified into 49 families on the basis of anticodon
specificity. There is only a rough correlation of human tRNA gene number with amino acid
frequency. For example, 30 tRNA gene specify the comparatively rare amino acid cystein
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(which accounts for 2.5% of all amino acids in human proteins), but only 21 tRNA genes
specify the more abundant proline (which has a frequency of 6.1%). Although the tRNA
genes seem to be dispersed throughout the human genome, more than half of human tRNA
genes (273 out of 516) reside on either chromosome 6 or 1. In addition, 18 of the 30 cys
tRNAs are found in a 0.5 Mb stretch of chromosome 7. tRNAs are adaptor molecules that
deliver amino acids to the ribosome and decode the information in mRNA. Their primary
structure (i.e. the linear sequence of nucleotides) is 60-95 nucleotide (nt) long, but most
commonly 76. They have many modified bases sometimes accounting 20% of the total bases
in any one tRNA molecule. Indeed, over 50 different types of modified base have been
observed in the tRNA molecules, and all of them are created post-transcriptionally. Four of
these, ribothimidine (T), which contains the base thymine not usually found in RNA,
pseudouridine (), dihydro-uridin (D) and inosine (I) are very common in nearly all tRNA, all
but the last being present in nearly all tRNA molecules in similar positions in the sequence.
Inosine is part of the anticodon in some tRNA, and can recognize 2 or 3 different bases
(wobble). All tRNAs have a common secondary structure, the cloverleaf structure. The stem
is called the amino acid acceptor stem. See the picture for further details on the secondary
structure of tRNAs. There are 9 hydrogen bonds that help form the 3-D structure of tRNA
molecules. tRNAs are joined to amino acids to become aminoacyl-tRNAs (charged tRNAs) in
a reaction called aminoacylation. Special enzymes called aminoacyl-tRNA synthetase carry
out the joining reaction which is extremely specific. An example for the nomenclature: the
amino acid leucine is linked to its tRNA called tRNALeu by the leucyl-tRNA synthetase
resulting in the generation of leucyl-tRNALeu.
NON-PROTEIN CODING RNAs SLIDES 7-18
SLIDE 7 A New RNA World RNA genes are transcribed parts of the genome that do not encode proteins,
hence their other name “non-coding RNAs (ncRNAs)”. Much of the attention paid to the human genes has
focused on protein-coding genes because they were long considered to be far the functionally most important
part of our genome. Non-coding RNA molecules have been so underappreciated that raw draft of human genome
sequences reported in 2001 contained no analyses at all of human RNA genes! RNA was seen to be important in
very early evolution (RNA World) but its functions were imagined to have been very largely overtaken by DNA
and proteins. In recent times, the vast majority of RNA molecules were imagined to serve as accessory
molecules in the making of proteins. The last few years have witnessed a revolution in our understanding of the
importance of RNA and, although the number of protein-encoding genes has been steadily revised downward
since draft human genome sequences were reported, the number of RNA genes is constantly being revised
upward. The tiny mitochondrial genome was always considered to exceptional because 65% (24 out of 37) of its
genes are RNA genes (22 tRNA and 2 rRNA genes). Now we are beginning to realize that the RNA transcribed
from the nucleus is not so uniformly dedicated to the protein synthesis as we once thought; instead, it shows
great functional diversity. What has changed our thinking? (1) First, completely unsuspected classes of ncRNAs
have recently been discovered. (2) Secondly, recent whole-genome analyses have shown that at least 85% and
possibly more than 90% of the human genome is transcribed. Two other major surprises were (3) the extent of
multigenic transcription, and (4) the pervasiveness of bidirectional transcription (about 70% of human genes are
transcribed from both strands; non-coding DNAs can be transcribed from both strands, too). The recent data
challenge the distinction between genes and intergenic space and have forced a radical rethink of the concept of
the gene. We have known for many decades that various ubiquitous ncRNA classes are essential for cell
function. Until recently, however, we have largely been accustomed to thinking of ncRNAs as not much more
than a series of accessories that are needed to process genes to make proteins. Transfer RNAs are needed at the
very end of the pathway, serving to decode the codons in mRNA and provide amino acids in the order they are
needed for insertion into the growing polypeptide chains. Ribosomal RNAs are essential components of the
ribosomes, the complex ribonucleoprotein factories of protein synthesis. Other ubiquitous ncRNAs were known
to function higher up the pathway to ensure correct processing of mRNA, tRNA and rRNA precursors. Various
small RNAs are components of complex ribonucleoproteins involved in different processing reactions, including
splicing, cleavage of rRNA and tRNA precursors, and base modifications that are required for RNA maturation.
Typically, these RNAs work as guide RNAs, by base pairing with complementary sequences in the precursor
RNA. We have also long been aware of a few ncRNAs that have other functions, such as RNAs implicated in X-
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inactivation and imprinting, and the RNA component of telomerase ribonucleoprotein needed for the synthesis of
the DNA telomeres. But these RNAs seemed to be quirky exceptions. In the past decade or so, however, there
has been a revolution in how we view RNAs. Many thousands of different ncRNAs have recently been
identified. Many of them are developmentally regulated and have been shown to have crucial roles in a whole
variety of different processes that occur in specialized tissues or different stages in development. Several
ncRNAs have already been implicated in cancer and genetic disease. May be it is time to view our genome as
more of an RNA machine than just a protein machine.
SLIDE 8 Ribozymes: relics of an ancient world? A ribozyme (from ribonucleic acid
enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule possessing a well
defined tertiary structure that enables it to catalyze a chemical reaction. Many natural
ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the
hydrolysis of bonds in other RNAs, but they have also been found to catalyze the
aminotransferase activity of the ribosome. RNAseP, for example, is a ribozyme that can
cleave substrate RNA without any requirement for proteins, and certain types of intron are
autocatalytic and able to splice themselves out of RNA transcripts without any help from
proteins. The peptidyl transferase activity - the enzyme that catalyzes the peptide bond – is a
ribozyme.
SLIDE 9 Small nuclear RNAs (snRNAs) Various families of rather small RNA molecules
(60 to 360 nucleotides long) are known to have a role in the nucleus in assisting general gene
expression, mostly at the level of post-transcriptional processing. Types: spliceosomal
snRNAs, non-spliceosomal snRNAs, small nucleolar RNAs (snoRNAs), small Cajal body
RNAs (scaRNAs).
Spliceosomal small nuclear RNA genes The nine human spliceosomal snRNAs vary in length from 106 to 186
nucleotides and bind a ring of seven core proteins. U1, U2, U4, U5, and U6 operate within the major
spliceosome to process conventional GU-AG introns. The other four spliceosomal snRNAs form part of the
minor spliceosome that excises AU-AC introns. More than 70 genes specify snRNAs used in the major
spliceosome. They include 44 identified genes specifying U6 snRNA and 16 specifying U1 snRNA.
Non-spliceosomal small nuclear RNA genes Not all snRNAs within the nucleoplasm function as part of
spliceosomes. Both U1 and U2 snRNAs also have non-spliceosomal functions.Us1 is required to stimulate
transcription elongation by RNA polymerase II. Several other snRNAs with a non-spliceosomal function tend to
be single-copy genes but there are many associated pseudogenes. Three examples are given bellow. (1) U7
snRNA is a 63-nucleotide RNA that is dedicated to the specialized 3’ processing undergone by histone mRNA
which, exceptionally, is not polyadenylated. (2) 7SK RNA is a 331-nucleotide RNA that functions as a negative
regulator of the RNA polymerase II elongation factor p-TEFb. (3) The Y RNA family consists of three small
RNAs that are involved in chromosomal DNA replication and function as regulators of cell proliferation.
Small nucleolar RNA (snoRNA) genes SnoRNAs are between 60 to 300 nucleotide long, and were initially
identified in the nucleolus, where they guide nucleotide modification in rRNA at specific positions. They do this
by forming short duplexes with a sequence of the rRNA that contains the target nucleotide. At least 340 human
snoRNA genes have been found so far, but there may be many more because snoRNAs are very difficult to
identify with the use of bioinformatics approaches. The vast majority is found within the introns of a larger gene,
which is transcribed by RNA pol-II. These snoRNAs are produced by processing of the intronic RNA, and so the
regulation of their synthesis is coupled to that of the host gene. Many snoRNAs genes are dispersed single-copy
genes. Others occur in clusters. Most snoRNAs are ubiquitously expressed, but some are tissue-specific.
Nonstandard functions are known or expected for some snoRNA genes that do not have sequences
complementary to rRNA sequences. For example, the HBII-52 snoRNA has an 18-nucleotide sequence that is
perfectly complementary to a sequence within the HTR2C (serotonin receptor 2c) gene at Xp24, and regulates
alternative splicing of this gene.
Small Cajal body RNA (scaRNA) genes Cajal bodies (CBs) are spherical sub-organelles of 0.3-1.0 µm in
diameter found in the nucleus of proliferative cells like embryonic cells and tumor cells, or metabolically active
cells like neurons. In contrast to cytoplasmic organelles, CBs lack any phospholipid membrane which would
separate their content, largely consisting of proteins and RNA, from the surrounding nucleoplasm. They were
first reported by Santiago Ramón y Cajal in 1903. The scaRNAs resemble snoRNAs and perform a similar role
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in RNA maturation, but their targets are spliceosomal snRNAs and they perform site-specific modifications of
spliceosomal snRNA precursors in the Cajal bodies of the nucleus. There are at least 25 human genes, each
specifying one type of scaRNA. Like snoRNA genes, the scaRNA genes are typically located within the introns
of genes transcribed by RNA pol-II.
ANTISENSE RNAs SLIDES 10-18
SLIDES 10, 11 Antisense RNAs
1. trans-antisense RNAs (imperfect homology): micro RNAs, siRNAs, piRNAs
2. cis-antisense RNAs (perfect homology): overlapping RNAs, siRNAs (?)
Cis position:
Trans position:
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close (overlapping) to the gene containing homologous sequences
far from the gene containing homologous sequences
SLIDES 12-14 MicroRNAs (miRNAs). A continuously increasing number of miRNAs have
been described in the genomes of several multicellular organisms. Micro RNA genes yield
RNA transcripts that are processed into short single-stranded segments, which then double
over on themselves to form hairpin structures. It has been proposed that they act as
components of protein/RNA complexes. A miRNA can both pair exactly with a mRNA and
cause its degradation via RNA interference (RNAi; see bellow) or it can pair partially with a
message and shut off translation. Recent studies involving computational approaches suggest
that the human genome may encode well over 1500 different miRNAs; the number known is
rising rapidly. A single micro RNA is assumed to regulate the expression of several genes. It
is hypothesized that up to one-third of human genes are regulated by these small RNAs. A
miRNA is a form of single-stranded (ss)RNS which is typically 20-25 nucleotides long. The
miRNAs are transcribed from DNA, but are not translated into protein. The DNA sequence
that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the
miRNA sequence and an approximate reverse complement. When this DNA sequence is
transcribed into a single-stranded RNA molecule, the miRNA sequence and its reversecomplement base pair to form a double stranded RNA hairpin loop; this forms a primary
miRNA structure (pri-miRNA). Drosha, a nuclear enzyme, cleaves the base of the hairpin to
form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus
into the cytoplasm. The Dicer enzyme then cuts 20-25 nucleotides from the base of the
hairpin to release the mature miRNA. The function of miRNAs appears to be in gene
regulation. For that purpose, a miRNA is complementary to a part of one or more mRNAs,
usually at a site in the 3’-UTR (untranslated region). The annealing of the miRNA to the
mRNA inhibits protein translation. In some cases, the formation of the double-stranded RNA
through the binding of the miRNA triggers the degradation of the mRNA transcript through a
process similar to RNAi, though in other cases it is believed that the miRNA complex blocks
the protein translation machinery or otherwise prevents protein translation without causing the
mRNA to be degraded.
Because many miRNAs are strongly conserved during evolution, vertebrate miRNAs were quickly identified.
miRNA regulate the expression of selected sets of target genes by base pairing with their transcripts. Usually, the
binding sites are in the 3’ untranslated region of the target mRNA sequences, and bound miRNA inhibits
translation so as to down-regulate expression of the target gene. Synthesis of miRNAs involves the cleavage of
RNA precursors by nuclease-specific and cytoplasm-specific RNA-III ribonucleases, nucleases that specifically
bind to and cleave double-stranded RNAs. The primary transcript, the pri-miRNA, has closely positioned
inverted repeats that base-pair to form a hairpin RNA that is initially cleaved from the primary transcript by a
nuclear RNase-III (known as Drosha) to make a short double-stranded pre-miRNA that is transported out of the
nucleus. A cytoplasmic RNAse III called Dicer cleaves the pre-miRNA to generate a miRNA duplex with
overhanging 3’ dinucleotides. A specific RNA-induced silencing complex (RISC) that contains the
endoribonuclease argonaute binds the miRNA duplex and acts to unwind the double-stranded miRNA. The
argonaute protein then degrades one of the RNA strands (the passenger strand) to leave the mature singlestranded miRNA (known as the guide strand) bound to argonaute. The mature miRNP (ribonucleoprotein)
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associates with RNA transcripts that have sequences complementary to the guide strand. The binding of miRNA
to target transcript normally involves a significant number of base mismatches. As a result, a typical miRNA can
silence the expression the expression of hundreds of target genes in much the same way that the tissue-specific
protein transcription factor can affect the expression of multiple target genes at the same time. Until now, more
than 700 human miRNA genes had been identified and experimentally validated, but comparative genomics
analyses indicate that the number of such genes is likely to increase. Some of the miRNA genes have their own
individual promoters; others are part of a miRNA cluster and are cleaved from a common multi-miRNA
transcription unit. Another class of miRNA genes form part of a compound transcription unit that is dedicated to
making other proteins in addition to miRNA, either another type of ncRNA or a protein.
SLIDE 15 Endogenous small interfering RNAs (endo-siRNAs) Long double-stranded RNA in mammalian
cells triggers nonspecific gene silencing through interferon pathways, but transfection of exogenous synthetic
siRNA duplexes or hairpin RNAs induces RNAi-mediated silencing of specific genes with sequence elements in
common with the exogenous RNA. Very recently, it has become clear that human cells also naturally produce
endo-siRNAs. Like piRNAs, endo-siRNAs are among the most varied RNA population in the cell (many tens of
thousands endo-siRNAs have been identified in mouse oocytes – itt kutatták). One way in which this happens
involves the occasional transcription of some pseudogenes.
SLIDE 16 Piwi-protein-interacting RNAs (piRNAs) have been found in a wide variety of eukaryotes. They
are expressed in germ-line cells in mammals and are typically 24-31 nucleotides long; they are thought to have a
major role in limiting transposition by retrotransposons, but they may also regulate gene expression. Control of
gene transposon activity is required because by integrating into new locations in the genome, active transposons
can interfere with gene function, causing genetic diseases and cancer. More than 15,000 different human
piRNAs have been identified and so the piRNA family is among the most diverse RNA family in human cells.
They are thought to be cleaved from large multigenic transcripts. The piRNAs are processed from long RNA
precursors transcribed from defined loci called piRNA clusters. Any transposon inserted in the reverse
orientation in the piRNA clusters can give rise to antisense piRNAs (shown in red). Antisense transposons are
incorporated into a piwi protein and direct its slicer activity on sense transposon transcripts. The 3’ cleavage
product is bound by another piwi protein and trimmed to piRNA size. The sense piRNA is, in turn, used to
cleave piRNA cluster transcripts and to generate more antisense piRNAs. Antisense piRNAs target the piwi
complexes to transcribing RNAs, which will lead to DNA methylation or histone modification (methylation of
histone) in the vicinity of the transcription.
SLIDES 17 Antisense overlapping RNAs Natural cis-encoded antisense RNAs are endogenous transcripts
that are transcribed from the opposite strand of the same genomic locus as the sense RNA and have a region of
perfect overlap with the sense transcripts. Very surprising novel data suggest that at least 30-40% of genes are
under the control of cis-antisense RNAs. The binding of mRNAs and antisense transcripts can sterically block
translation from mRNA or, alternatively, it may trigger the RNA interference pathway, which eventually leads to
the degradation of mRNA. Many thousands of different long ncRNAs, often many kilobases in length, are also
thought to have regulatory roles in animal cells. They include antisense transcripts that are usually do not
undergo splicing and that can regulate overlapping sense transcripts, plus a wide variety of long mRNA-like
ncRNAs that undergo splicing, and polyadenylation but do not seem to encode any sizable polypeptide, although
some contain internal ncRNAs such as snoRNAs and piRNAs. The functions of the great majority of the mRNAlike ncRNAs are unknown. Some, however, are known to be tissue-specific and involved in gene regulation. The
XIST gene encodes a long ncRNA that regulates X-chromosome inactivation, the process by which one of the
two X chromosomes is randomly selected to be condensed in female mammals, with large regions becoming
transcriptionally inactive (see lecture Epigenetics). Many other long ncRNAs, such as the H19 RNA, are
implicated in repressing the transcription of either paternal or maternal allele of autosomal regions (imprinting,
see lecture Epigenetics).
Role in the medicine The double stranded (ds)RNAs that trigger RNAi may be usable as drugs. Another
speculative use of dsRNA is in the repression of essential genes in eukaryotic human pathogens or viruses that
are dissimilar from any human genes; this would be analogous to how existing drugs work. RNAi interferes with
the translation process of gene expression and appears not to interact with the DNA itself. Proponents of
therapies based on RNAi suggest that the lack of interaction with DNA may alleviate some patients' concerns
about alteration of their DNA (as practiced in gene therapy), and suggest that this method of treatment would
likely be no more feared than taking any prescription drug. For this reason RNAi and therapies based on RNAi
have attracted much interest in the pharmaceutical and biotech industries.
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SLIDE 18 HAR (human accelerated regions) In the non-coding part of human genome it was described 49
region (HAR: human accelerated region), which were found highly conserved (unchanged) in vertebrate species,
but rapidly evolved (changed the nucleotide content) in human. Twelve out of 49 HARs are expressed in the
brain. The most rapidly changing among the HARs is the HAR1: in a 118 by DNA stretch, there is only 2basepair difference between chimp and chicken, while 18-base pair difference between chimp and human. It is
interesting that HAR1 is expressed in human brain cortex at 7-17th gestation period, which raise the question as
to whether these regions played roles in human evolution.
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The pre-initiation complex The pre-initiation complex facilitates the binding of RNA
polymerase II to the promoter of genes, which in turn initializes transcription. The RNA
polymerase II is composed of 12 subunits. RNA polymerase binding the promoter has to be
preceded by the attachment of several transcription factors to the promoter or to the
polymerase itself. The pre-initiation complex can only initiate a basal expression level from a
specific gene. Other elements at more distant position are needed for elevated expression
level.
mRNA transport Eukaryotic mRNAs must leave the nucleus in order to be translated into
proteins. Mature mRNAs exit through the nuclear pores, but the underlying mechanisms are
not fully understood. A large portion of unprocessed transcripts never leave the nucleus and
are degraded. Proteins traveling to the appropriate organelles are directed by the signal
peptides locating on their N-terminals. Another possibility for pass a certain protein to the
desired organelle is based on mRNA targeting. Some mRNAs contain a zip code on the 5’
termini, which contains information for the subcellular targeting of mRNA.
Texts written in small letters belong to the extra requirements
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