Translational genomics
Genome architecture
The human genome
Genetic information in humans is mainly stored in the nucleus, where it is organized into 22
pairs of autosomes and two sex chromosomes (XX or XY). In addition, a small circular
genome is present in the mitochondria. Together, the genome encodes ~20,000
protein-coding genes and ~25,000 non-coding genes, indicating the importance of
RNA-mediated regulation.
A difficulty in genome sequencing is the first exon of genes, which is often GC-rich. GC-rich
DNA is hard to denature because G–C pairs form three hydrogen bonds. This high GC
content ensures correct binding of the ribosome to mRNA and accurate initiation of
translation at the proper start codon (ATG)
Functional DNA
Functional DNA consists of:
- Protein coding genes.
- Non-coding genes.
- Regulatory elements.
The functional regions of protein coding genes are:
- Promoter: regulatory sequence upstream of
the TSS.
- 5′ UTR: untranslated regulatory region.
- Exons: protein encoding segments.
- Introns: non-coding segments removed by
splicing.
- 3′ UTR: untranslated, regulatory functions in
stability/translation.
- Intergenic regions: space between genes,
often containing enhancers/silencers.
Clinical sequencing covers only the 5′ UTR – 3′ UTR region because mutations in the coding
region are often directly linked to a disease. Promoters are excluded because variants are
harder to interpret; if promoter mutations are suspected, more specialized tests are required.
Alternative splicing: Different exon combinations result in multiple isoforms from one gene,
this expands the proteome and functional diversity.
,Non-coding genes
A large portion of the genome encodes RNAs that do not code for proteins, but regulate
gene expression.
Non-Coding RNAs: ncRNAs. Functional RNA molecules that are not translated and have
regulatory functions. Deregulation of these genes results in various disorders.
Classification
- Small ncRNAs (~20–25 nt): miRNA, siRNA, piRNA.
- Long ncRNAs (lncRNAs; 200–3000 nt).
Small ncRNAs
MicroRNAs (miRNAs): ~21–22 nucleotides. Regulate post-transcriptional gene
expression.
Biogenesis
1. Transcribed by RNA polymerase II →
pri-miRNA (hairpin structure).
2. In nucleus: Drosha/Pasha complex
cuts pri-miRNA → pre-miRNA.
3. Exported cytoplasm by Exportin-5.
4. Dicer removes hairpin loop → short
dsRNA.
5. Incorporated into RISC (RNA-induced
silencing complex) with Argonaute
(AGO). One strand is degraded; the
other serves as guide sequence.
miRNA function: Guides the RISC complex
to complementary target mRNA sequences
(usually at the 3′UTR). This results in:
- Inhibition of translation initiation.
- Inhibition of elongation.
- Deadenylation of the polyA-tail and
degradation of target mRNA.
One miRNA can target many mRNAs and
multiple miRNAs can regulate a single
transcript.
Disease relevance: Feingold syndrome type 1: loss of miR-17~92 cluster.
Polycistronic gene: contains multiple coding genes.
,Small interfering RNAs (siRNAs): ~21–22 nts, double-stranded RNA molecules. Derived
from endogenous or exogenous long dsRNA. They function primarily in RNA interference
(RNAi), a highly specific gene-silencing mechanism.
- Exogenous siRNAs: introduced from outside (e.g. viral RNA, experimental
transfection, therapeutic siRNA).
- Endogenous siRNAs: generated inside cells from longer dsRNA precursors (can
arise from transposons, repetitive sequences, etc).
Biogenesis
1. Dicer processing: dsRNA is recognized by the
RNase III enzyme Dicer, which cleaves it into
short ~21–22 nt siRNA duplexes.
2. RISC loading: The siRNA duplex is loaded into
the RISC, which also contains AGO proteins.
3. The guide strand is retained in RISC; the
passenger strand is degraded.
siRNA function: The siRNA guide strand directs RISC to
its target mRNA by perfect Watson–Crick base pairing.
AGO2 then cleaves the target mRNA at the site of
pairing. This marks the mRNA for degradation, leading
to near-complete silencing of the gene.
miRNAs vs siRNAs
miRNAs usually have imperfect complementarity to their targets, leading to translation
inhibition or destabilisation. siRNAs, in contrast, usually show perfect complementarity,
resulting in the direct cleavage of target mRNA.
Targets:
- siRNAs: often transposons, viral RNAs, repetitive sequences, or exogenous RNAs.
- miRNAs: mainly endogenous protein-coding genes.
Transposon silencing: siRNAs prevent genomic instability by inactivating transposable
elements.
Experimental and therapeutic applications:
- Researchers use synthetic siRNAs to knock down target genes in cell systems.
PIWI-interacting RNAs (piRNAs): 24–31 nts. They do not require Dicer; processed from
single-stranded precursors. They are expressed mainly in germ cells.
Function: silence transposable elements during spermatogenesis & oogenesis, protecting
genome integrity.
Largely irrelevant for regular somatic functions.
, Long ncRNAs
Long Non-Coding RNAs (lncRNAs): ~200–3000 nucleotides. Widespread in the genome,
highly diverse.
Genomic Origin & Classification
- Intronic lncRNAs: entirely transcribed from an intron of a protein-coding gene.
Usually close to their locus and can regulate the expression or splicing of the host
gene.
- Intergenic lncRNAs (lincRNAs): found in the “intergenic” regions, independent of
protein-coding sequences. Can act at long distances to regulate multiple genes.
- Natural Antisense Transcripts (NATs): produced from the DNA strand opposite to a
coding or non-coding gene. Because they are complementary, they can bind the
sense mRNA and regulate its:
- Stability (protection from degradation or targeting RNases).
- Splicing (masking splice sites).
- Translation (blocking ribosome binding).
- NATs can also recruit chromatin remodelers at the locus, controlling
transcription in an allele-specific manner.
MYCN-OS: NAT on the opposite strand. Regulates the other strand. Feingold
syndrome Type2! NAT mechanisms of action.
Mechanisms of Action
lncRNAs are extremely versatile. Their regulatory functions generally fall into 4 main modes:
1. Signal: expression of lncRNA occurs at a specific time/place as a marker of
transcriptional state.
2. Decoy: lncRNA binds to and confiscates proteins or RNAs, preventing them from
acting on their target, acting as a “sponge” for transcription factors or miRNAs.
3. Guide: lncRNA binds epigenetic-modifier proteins (e.g. histone methyltransferases)
and guides them to specific genomic loci in cis (nearby gene) or in trans (far-away
gene).
4. Scaffold: lncRNA acts as a platform to assemble multiple proteins into a functional
complex, e.g. scaffolding chromatin-modifying complexes or transcription machinery
components.
Genome architecture
The human genome
Genetic information in humans is mainly stored in the nucleus, where it is organized into 22
pairs of autosomes and two sex chromosomes (XX or XY). In addition, a small circular
genome is present in the mitochondria. Together, the genome encodes ~20,000
protein-coding genes and ~25,000 non-coding genes, indicating the importance of
RNA-mediated regulation.
A difficulty in genome sequencing is the first exon of genes, which is often GC-rich. GC-rich
DNA is hard to denature because G–C pairs form three hydrogen bonds. This high GC
content ensures correct binding of the ribosome to mRNA and accurate initiation of
translation at the proper start codon (ATG)
Functional DNA
Functional DNA consists of:
- Protein coding genes.
- Non-coding genes.
- Regulatory elements.
The functional regions of protein coding genes are:
- Promoter: regulatory sequence upstream of
the TSS.
- 5′ UTR: untranslated regulatory region.
- Exons: protein encoding segments.
- Introns: non-coding segments removed by
splicing.
- 3′ UTR: untranslated, regulatory functions in
stability/translation.
- Intergenic regions: space between genes,
often containing enhancers/silencers.
Clinical sequencing covers only the 5′ UTR – 3′ UTR region because mutations in the coding
region are often directly linked to a disease. Promoters are excluded because variants are
harder to interpret; if promoter mutations are suspected, more specialized tests are required.
Alternative splicing: Different exon combinations result in multiple isoforms from one gene,
this expands the proteome and functional diversity.
,Non-coding genes
A large portion of the genome encodes RNAs that do not code for proteins, but regulate
gene expression.
Non-Coding RNAs: ncRNAs. Functional RNA molecules that are not translated and have
regulatory functions. Deregulation of these genes results in various disorders.
Classification
- Small ncRNAs (~20–25 nt): miRNA, siRNA, piRNA.
- Long ncRNAs (lncRNAs; 200–3000 nt).
Small ncRNAs
MicroRNAs (miRNAs): ~21–22 nucleotides. Regulate post-transcriptional gene
expression.
Biogenesis
1. Transcribed by RNA polymerase II →
pri-miRNA (hairpin structure).
2. In nucleus: Drosha/Pasha complex
cuts pri-miRNA → pre-miRNA.
3. Exported cytoplasm by Exportin-5.
4. Dicer removes hairpin loop → short
dsRNA.
5. Incorporated into RISC (RNA-induced
silencing complex) with Argonaute
(AGO). One strand is degraded; the
other serves as guide sequence.
miRNA function: Guides the RISC complex
to complementary target mRNA sequences
(usually at the 3′UTR). This results in:
- Inhibition of translation initiation.
- Inhibition of elongation.
- Deadenylation of the polyA-tail and
degradation of target mRNA.
One miRNA can target many mRNAs and
multiple miRNAs can regulate a single
transcript.
Disease relevance: Feingold syndrome type 1: loss of miR-17~92 cluster.
Polycistronic gene: contains multiple coding genes.
,Small interfering RNAs (siRNAs): ~21–22 nts, double-stranded RNA molecules. Derived
from endogenous or exogenous long dsRNA. They function primarily in RNA interference
(RNAi), a highly specific gene-silencing mechanism.
- Exogenous siRNAs: introduced from outside (e.g. viral RNA, experimental
transfection, therapeutic siRNA).
- Endogenous siRNAs: generated inside cells from longer dsRNA precursors (can
arise from transposons, repetitive sequences, etc).
Biogenesis
1. Dicer processing: dsRNA is recognized by the
RNase III enzyme Dicer, which cleaves it into
short ~21–22 nt siRNA duplexes.
2. RISC loading: The siRNA duplex is loaded into
the RISC, which also contains AGO proteins.
3. The guide strand is retained in RISC; the
passenger strand is degraded.
siRNA function: The siRNA guide strand directs RISC to
its target mRNA by perfect Watson–Crick base pairing.
AGO2 then cleaves the target mRNA at the site of
pairing. This marks the mRNA for degradation, leading
to near-complete silencing of the gene.
miRNAs vs siRNAs
miRNAs usually have imperfect complementarity to their targets, leading to translation
inhibition or destabilisation. siRNAs, in contrast, usually show perfect complementarity,
resulting in the direct cleavage of target mRNA.
Targets:
- siRNAs: often transposons, viral RNAs, repetitive sequences, or exogenous RNAs.
- miRNAs: mainly endogenous protein-coding genes.
Transposon silencing: siRNAs prevent genomic instability by inactivating transposable
elements.
Experimental and therapeutic applications:
- Researchers use synthetic siRNAs to knock down target genes in cell systems.
PIWI-interacting RNAs (piRNAs): 24–31 nts. They do not require Dicer; processed from
single-stranded precursors. They are expressed mainly in germ cells.
Function: silence transposable elements during spermatogenesis & oogenesis, protecting
genome integrity.
Largely irrelevant for regular somatic functions.
, Long ncRNAs
Long Non-Coding RNAs (lncRNAs): ~200–3000 nucleotides. Widespread in the genome,
highly diverse.
Genomic Origin & Classification
- Intronic lncRNAs: entirely transcribed from an intron of a protein-coding gene.
Usually close to their locus and can regulate the expression or splicing of the host
gene.
- Intergenic lncRNAs (lincRNAs): found in the “intergenic” regions, independent of
protein-coding sequences. Can act at long distances to regulate multiple genes.
- Natural Antisense Transcripts (NATs): produced from the DNA strand opposite to a
coding or non-coding gene. Because they are complementary, they can bind the
sense mRNA and regulate its:
- Stability (protection from degradation or targeting RNases).
- Splicing (masking splice sites).
- Translation (blocking ribosome binding).
- NATs can also recruit chromatin remodelers at the locus, controlling
transcription in an allele-specific manner.
MYCN-OS: NAT on the opposite strand. Regulates the other strand. Feingold
syndrome Type2! NAT mechanisms of action.
Mechanisms of Action
lncRNAs are extremely versatile. Their regulatory functions generally fall into 4 main modes:
1. Signal: expression of lncRNA occurs at a specific time/place as a marker of
transcriptional state.
2. Decoy: lncRNA binds to and confiscates proteins or RNAs, preventing them from
acting on their target, acting as a “sponge” for transcription factors or miRNAs.
3. Guide: lncRNA binds epigenetic-modifier proteins (e.g. histone methyltransferases)
and guides them to specific genomic loci in cis (nearby gene) or in trans (far-away
gene).
4. Scaffold: lncRNA acts as a platform to assemble multiple proteins into a functional
complex, e.g. scaffolding chromatin-modifying complexes or transcription machinery
components.
