Transcript RNA function + maturation - Wei Li Lab

Exploring your RNA World with an
Emphasis on Non-coding Transcripts
Benjamin Rodriguez, PhD
Wei Li Lab, Baylor College of Medicine
Molecular Biology Refresher Course with Bioinformatics
September 13th 2013
Software, Sites, Materials
Course Materials:
http://dldcc-web.brc.bcm.edu/lilab/benji/MBRB_2013/index.html
Most up to date slides
I will upload for all three of my lectures
Browsers:
http://genome.ucsc.edu/
http://epigenomegateway.wustl.edu/
Web-based analysis:
http://mirtar.mbc.nctu.edu.tw/human/index.php
http://lilab.research.bcm.edu/cpat/
Outline
• Types of RNA transcripts and their functions
• Post-transcriptional regulation by microRNAs
– Mouse models and clinical efficacy
• Noncoding RNAs and genomic imprinting
– CDKN1C and Kcnqot1
• Lab exercises: microRNA target prediction and
assessing protein coding potential of novel
transcripts
It’s an RNA World, Baby!
• Protein Translation
• mRNA
• tRNA
• rRNA
• RNA function and maturation
• snRNA
• snoRNA
• RNaseP
• Y RNA
• Rnase MRP
• RNA interference
• miRNA
• siRNA
• piRNA
• Regulatory RNAs
• lncRNA
• lincRNA
• Telomere synthesis
• Telomerase RNA
Functions of RNAs
• Protein Translation
• mRNA
• tRNA
• rRNA
Protein translation
Messenger RNA (mRNA)
• Destiny dictated by post-transcriptional modifications
• “Cap and tail exits cell”
• mRNA methylation widespread and likely functional
• N6-methyladenosine (m6a)
• meRIP-Seq
Protein translation
Transfer RNA (tRNA)
• 15% of cellular RNA
Ribosomal RNA (rRNA)
• 80% of cellular RNA
Functions of RNAs
•RNA function + maturation
• snRNA
• snoRNA
• RNaseP
• Y RNA
• Rnase MRP
RNA-protein complexes
Support cellular and
molecular functions
RNA function and maturation
Small nuclear RNA (snRNA)
• RNA component of the Spliceosome
• snRNP complex made up of 5 snRNAs and over 20 proteins
• Removes regions of non-coding mRNA (introns)
RNA function and maturation
Small nucleolar RNA (snoRNA)
• Guides chemical modifications of other RNAs
• mainly rRNA, tRNA, and snRNA
• 2 main classes of snoRNA:
• H/ACA box, direct conversion of uridine to pseudouridine
• C/D box snoRNAs, help add methyl groups to RNAs
RNA function and maturation
Ribonuclease P (RNaseP)
• RNA component of an RNA enzyme (Ribozyme)
• Cleaves a precursor sequence from tRNA molecules, generating
mature tRNA
• Also required for RNA Pol III transcription of various small noncoding RNA
genes (e.g., tRNA, 5S rRNA, SRP RNA, and U6 snRNA genes)
• Because they catalyze site-specific cleavage of RNA molecules, Ribozymes
may have pharmaceutical applications
RNA function and maturation
Y RNA
• Component of Ro Ribonucleoparticle (RoRNP) complex
• Chaperone regulating maturation of small ncRNAs
• Transcribed by RNA Pol III
• UV resistance in mammalian cells
• Essential for DNA replication
• Upregulated in human cancer tissue
• Required for increased proliferation of cancer cell lines
RNA function and maturation
Ribonuclease MRP (RNase MRP)
• RNA component of RNase MRP
• Enzymatically active ribonucleoprotein
• Initiation of mitochondrial DNA replication
• In the nucleus, involved in precursor rRNA
processing
Functions of RNAs
• RNA interference
• miRNA
• siRNA
• piRNA
RNA inhibiting RNA
RNA interference (RNAi)
• Biological process in which RNA molecules inhibit gene expression,
typically by causing the destruction of specific mRNA molecules
• Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Medicine
for their work on RNAi in the nematode worm C. elegans
• RNAi helps defend cells against parasitic nucleotide sequences
• viruses and transposons
• Plays integral role in development as well as regulation of gene
expression in general
• Technological applications
• Gene knockdown: study physiological role of individual genes
• Functional genomics: genome-wide RNAi screens
• Medicine: attractive, but RNAi delivery to tissues is difficult
Small (short) interfering RNA (siRNA)
MicroRNA (miRNA)
miRNAs must first undergo extensive post-transcriptional
modification before they are mature and functional
Primary transcript
(Pri-miRNA)
pre-miRNA
mature miRNA
1. Pri-miRNA are processed into 70-nucleotide precursors (pre-miRNA)
2. Precursor is cleaved to generate 21–25-nucleotide mature miRNAs
miRNA processing
Step 1
• Pri-miRNA transcript is processed by RNAse III
enzyme Drosha and dsRNA-binding protein DGCR8
• Drosha cleaves these into 70-bp pre-miRNAs that
consist of an imperfect stem-loop structure
miRNA processing
Step 2
• Pre-miRNA exported from the nucleus into the cytoplasm by
Exportin 5 (Exp5)
• Cleavage by Dicer yields small, imperfect dsRNA duplex
(miRNA: miRNA*) containing both mature miRNA strand and
its complementary strand
miRNA meet RISC
RNA Induced Silencing Complex (RISC)
• Thermodynamics properties may dictate which strand is
incorporated into RISC
• miRNA-RISC complex bind to 3’ UTRs of their targets
• May repress transcription through RNA cleavage or
repress further translation of nascent protein peptides
• Method of repression may depend on extent of
sequence complementarity between miRNA and target
miRNAs and cancer
• Mouse models implicate the gain or loss of individual
microRNAs (miRNAs), miRNA clusters and the miRNA
processing machinery in cancer
• Oncogenic
• miR-155, miR-21 and miR-17~92
• Tumor-suppressive
• miR-15~16, LIN28, DICER
• Context-dependent
• miR-146 and miR-29
• miRNAs and miRNA processing machinery involved in all
stages of metastatic disease
• Studies uncovering miRNA function have led to their
therapeutic application
Mouse models to evaluate in vivo miRNA functions
Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166
Opposing roles of miRNAs in cancer
Oncogenic versus tumor-suppressive functions of miR-146 can be
explained based on upstream nuclear factor κB (NF-κB) signals
Opposing roles of miRNAs in cancer
Oncogenic miR-146 also targets BRCA1, thus preventing the proapoptotic effects of BRCA1 and resulting in a pro-survival response
Opposing roles of miRNAs in cancer
Tumor-suppressive miR-29a targets multiple oncogenes, inhibiting
growth and proliferation as well as aggressive disease (B-CLL)
Opposing roles of miRNAs in cancer
Oncogenic miR-29a prevents cell adhesion through repressing
peroxidasin homologue (PXDN)
Therapeutics: replacing tumor-suppressor miRNAs
• let-7 downregulated in multiple cancers
• Prime example of therapeutic efficacy
Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166
Therapeutics: some miRs best left alone
• miR-122 plays pleiotropic role in multiple pathophysiologies
• Administration may reduce HCC metastasis
• Antagomir decreases HCV viraemia (primates) and
cholesterol levels (mouse)
Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166
Therapeutics: replacing tumor-suppressor miRNAs
• miR-26a administration inhibited HCC proliferation, induced
apoptosis and protected animals from disease progression
Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166
Therapeutics: targeting oncogenic miRNAs
• Antagomir-miR-10b treatment had no effect on the primary
breast tumor xenograft, but reduced pulmonary metastases
by more than 80 %
Nature Reviews Cancer 11, 849-864 (December 2011) | doi:10.1038/nrc3166
miR-29b: targeting a predictor of
treatment response in AML
• Rational, combinatorial targeting multiple of multiple
proteins in a leukemogenic pathway with a cocktail of
pharmacologic inhibitors
• miR-29b targets DNMTs, thereby resulting in global DNA
hypomethylation and reexpression of hypermethylated,
silenced genes in AML
• Higher baseline expression of miR-29b associated with clinical
response (P = 0.02) in a phase II decitabine trial of previously
untreated adult AML patients
• Proteasome inhibitor bortezomib increases expression of
miR-29b and decreases leukemia cell growth
• Phase I study of DAC and bortezomib in adults with relapsed
or refractory AML (Aug 2013)
• Five of 23 patients achieved remission (CR and/or Cri)
Telomere synthesis
Telomerase RNA
• Telomerase extends ends of DNA molecules
• preventing DNA loss during replication
• Protein component possesses reverse transcriptase activity
• RNA component serves as a template for the telomere repeat
• Telomerase dysregulation is a hallmark of cancer
Regulatory RNAs
Long non-coding RNA (lncRNA, lincRNA)
• Non-protein coding transcripts 200 nt to 100 kb in length.
• lncRNAs characterized thus far appear to function in diverse areas
including epigenetics, alternative splicing, and nuclear import
• XIST plays a critical role in X-chromosome inactivation
• Kcnq1ot1 ncRNA is required for the maintenance of the silencing of
ubiquitously imprinted genes (UIGs) at all developmental stages
Remember Genomic Imprinting?
Imprinted genes occur in clusters and are often involved
in growth control
Maternal allele
Paternal allele
Paternal allele
Maternal allele
Imprinting regulated by cis-acting elements (Imprinting
Control Regions) and noncoding RNAs They act over long
distances and control the imprinting of multiple genes
CDKN1C: Regulator of Embryonic Growth
• CDKN1C is required for postnatal survival
• Altered cell differentiation and proliferation in
mice lacking CDKN1C
• Developmental defects consistent with a
causative role for in Beckwith-Wiedemann
syndrome
+/+
+/-
•Skeletal abnormalities
•Abdominal muscle defects
•Adrenomegaly
-/-
Nature 1997; 387(6629): 151-158
CDKN1C/KCNQ1OT1 Imprinted Domain
Unmethylated paternal allele functions as a silencer and
a promoter for KCNQ1OT1 transcription
Methylated maternal allele cannot function as a silencer
or a promoter for KCNQ1OT1 and thus expresses
CDKN1C
Antisense Transcripts are involved in
Genomic Imprinting
• Linked to mechanisms of monoallelic gene
expression, including genomic imprinting and X
chromosome inactivation
• RNAs with sequence complementarity to other
transcripts
• May regulate expression of target genes at level
of transcription, mRNA processing, or translation
Sense Gene
Antisense
transcript
Discovery of a paired CDKN1C antisense transcript
The CDKN1C-AS transcript aligns in cis to the CDKN1C promoter
and gene body, spanning at least 2.5 kb in length.
E2 induces antisense CDKN1C expression in epigenetically
reprogrammed breast cancer cells
CDKN1C-AS significantly induced by 12 hr E2 treatment in MCF7
cells subjected to pharmacologic inhibition of DNA methylation
and histone deacetylation (P < 0.01)
Assessing CDKN1C-AS function
p57-S: CDKN1C promoter drives transcription of GFP
p57-AS: CMV promoter drives CDKN1C-AS transcription
p57-ASΔ: contains a stop sequence downstream of CMV
promoter that prevents CDKN1C-AS transcription
Assessing CDKN1C-AS function
Exogenous antisense transcript failed to
repress GFP expression in cis
Assessing CDKN1C-AS function
Exogenous antisense expression induced > 35-fold
Assessing CDKN1C-AS function
**
NS
CDKN1C-AS represses endogenous CDKN1C in trans
Repression ameliorated by p57-ASΔ
Proposed regulatory mechanism of CDKN1C-AS
Formation of double-stranded RNA may negatively
regulate stability, transport, and/or translation of sense
CDKN1C transcript
E2 induces expression of KCNQ1OT1 ncRNA
Expression of KCNQ1OT1 increased 1.6-fold
following 12 hr E2 treatment (P < 0.005)
E2 induces recruitment of transcription regulator proteins to
the 11p15.5 ICR
Fraction of Input
HDAC1
*
RNA Pol II
Fraction of Input
CTCF
*
IgG
Vehicle
E2
Epigenetic repression of CDKN1C through coordinated loop
formation with the 11p15.5 ICR
• Binding of CTCF mediates long range epigenetic
silencing through formation of chromatin loop
structures
• 11p15.5 ICR recently shown to physically interact
with the CDKN1C promoter in MCF7 cells
• Estrogen signaling can trigger complex loop
formation between cis-regulatory regions and
promoters
Epigenetic repression of CDKN1C through coordinated loop
formation with the 11p15.5 ICR
CTCF binding to ICR and CDKN1C locus
forms a long-range intrachromosomal
loop via dimerization of CTCF
Ligand-bound ERα complex may mediate silencing
through a secondary loop
Epigenetic repression of CDKN1C through coordinated loop
formation with the 11p15.5 ICR
CTCF serves as scaffold to secure PRC2
complex which methylates H3K27,
leading to formation of a repressive
chromatin state
ERα-mediated loop
sequesters upstream tissuespecific enhancers
Recruits PRC2 and HDAC1
Summary
• RNA transcripts perform a diverse set of
functions in the cell independent of coding
proteins
• MicroRNAs are promising therapeutic targets
• Noncoding RNAs play an important role in
epigenetic mechanisms of transcriptional
silencing
• Lab exercises: microRNA target prediction and
assessing protein coding potential of novel
transcripts
Laboratory Exercises
• microRNA target prediction
• http://mirtar.mbc.nctu.edu.tw/human/index.php
• Assessing protein coding potential of novel
transcripts
• http://lilab.research.bcm.edu/cpat/
microRNA target prediction
http://mirtar.mbc.nctu.edu.tw/human/index.php
• miRTar is an actively updated web-based program
• Flexible, easy to use interface
• Experimentally
In this example, we search for
miR-148a targets sites in the
3’ UTR of the DNMT3B gene
microRNA target prediction
• After submitting job, GUI presents input parameters
• Target linkage between microRNA and gene is represented in
separate hyperlinks which retrieve different information
miRNA icon takes the user to mirBase, a
database of multiple-species microRNA
sequence, target, and annotation
information
microRNA target prediction
Gene icon provides detailed information on the gene, miRNA targets on
transcripts (mRNA records corresponding to the searched gene), and an
overlay of potential alternative splicing events
Target metrics, graphical and sequence representation
microRNA target prediction
• This graphical representation connects transcript to protein
information on a single screen window
• Searching for the various
targets of multiple
microRNAs
• We searched 10 miRNAs
versus 69 genes
• We searched 10 miRNAs
versus 69 genes
Choose KEGG to analyze pathway information for let-7a
predicted target genes
Kyoto Encyclopedia of Genes
and Genomes
Bioinformatics resource for
linking genomes to life and
the environment
Pathway enrichment analysis for let-7a-predicted targets
• Tired: no multiple-testing correction
• Searching for targets of
miR-29b which belong to
the TGF-beta signaling
pathway
• Produces a summary
table with hyperlinks
• Let us analyze KEGG and
see if TGF-beta is the top
result?
• As you can see, your starting list is the most highly
enriched result
• program should probably warning you about this
• Lists are not necessarily independent of one another
Click on the TGF-beta hyperlink to bring up a
KEGG pathway map
• Diagram well summarizes lots of complex information
Hyperlinks allow you to access individual gene results, similar to
other modes
Can upload files of sequences or URLs
Select example sequence in FASTA
Set species to Human
Choose submit
CPAT Output
Coding probability predicted to be > 99 %
Let’s test mouse Kcnq1ot1!
Sequence pasted as BED or FASTA data
Coding probability predicted to be < 12 %
Our analysis further suggests mouse Kcnq1ot1 is a noncoding transcript