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The ribosomopathy paradox

By K. Sri Manjari
Research Scholar
Department of Cell Biology
Institute of Genetics & Hospital for Genetic Diseases

Molecular Biology Begins With

DNA, Codons and the Amino Acid


Code

Collectively, the ribosomopathies are


caused by defects in ribosome
biogenesis. Although these disorders
encompass deficiencies in a ubiquitous
and fundamental process, the clinical
manifestations
are extremely variable and typically
display tissue specificity.

DBA is a
ribosomopathy

Mutations affecting
ribosomal protein
(RP) genes

DBA
Mutations affecting:

25-35%

25%
RPS19

RPL5, RPL11, RPS26,


RPS24, RPS17, RPS10,
RPL35a,RPS7, RPL26,
RPL15

40-50% ??

Heterozygous,
autosomal
dominant

~80 RP genes in total


10 are known to be affected in DBA
GATA1 may also have a role

Leading to RP
haploinsufficiency

DNA, RNA & Proteins (& Cake)

Amino acids

The Ribosome (80S)


60S (L) unit:
5S RNA,
28S RNA,
5.8S RNA +
~49 proteins

40S (S) unit:


18S RNA +
33 proteins
A cake making machine that uses mRNA as the recipe and amino acids as the
ingredients

Types of Mutations in DBA 1)


Missense

Change in recipe use salt instead of sugar


= cake no good!

Types of Mutations 2) Nonsense

Change in recipe leave out half of ingredients


= cake no good!

Types of Mutations 3) Frameshift

Change in recipe words become unreadable


= cake no good!

Types of Mutations 4) Splice Site

Change in recipe pages left out or go blank


= cake no good!

Types of Mutations 5) Copy Number


Variation (CNV)

Change in recipe pages torn out


= cake no good!

10 Commonly Identified DBA associated RP Genes


Mutations are mostly SNVs and indels, but large deletions & insertion are also seen
RPS26

RPS24

RPS7

RPS17

RPL26

RPS35a
RPL11
RPS10

Unknown

RPL5

RPS19

= 7 genes in conventional molecular screen

Standard
DBA
Screening
Pipeline

Measure & QC
Peripheral Blood
Extract DNA

RPS19
RPL5
RPL11
RPS24
RPS17
RPL35a
RPS7

Sanger Sequence

PCR target gene exons

Genes: Exons, Introns & Splicing

For many years now, gene expression


has been measured as a
reflection of transcriptional activation,
and the assumption has
been made that the absolute level of
mRNA for a given gene
within the cell directly correlates with
protein level for that
gene.

Although mRNA level strongly


correlates with protein
expression, more recent evidence
highlights the very important
role that posttranscriptional events,
including translation and microRNA
(miRNA) regulation of mRNA, play in
regulating gene
expression

Similar to key regulators of gene


transcription (e.g., p53
or c-Myc), key regulators of translation
are specifically targeted
in human diseases, including cancer.

Indeed, recent data suggest


that RNA binding proteins (RBPs) are frequently
associated with disease
Fragile-X mental
retardation protein

Fragile-X syndrome and


autism

Darnell et al.,
2011

musashi-1 and -2

Stem cell biology and


leukemia

Kharas et al., 2010

NPM1

hematological
malignancies

Grisendi et al., 2006

RPS7, RPS10,
RPS17, RPS19

DiamondBlackfan
anemia

Idol et al, 2009

TCOF1, POLR1D,
POLR1C

Treacher Collins
syndrome

Dauwerse et al, 2011

DKC1

dyskeratosis congenita

Heiss et al, 2002

THE PARADOX..
Individually, the ribosomopathies are rare and
phenotypically unique.
Intuitively, mutations affecting the ribosome, a
molecule essential for protein synthesis in every
cell, should affect all tissues and cell types.
On the contrary, ribosome biogenesis disorders
are highly heterogeneous in both their physical
manifestations and modes of inheritance, and
there is a surprising tendency toward tissue
specificity in these diseases

How can ribosomopathies first appear


as diseases caused by too few cells,
but later turn into diseases caused by
too many cells? This paradox has
puzzled the scientific community for
years.

Over the last number of years, there has been increasing awareness of
the role that ribosome, ribosome biogenesis, and various other factors
that relate to translation play in normal cellular homeostasis, and in
human disease (Xue and Barna, 2012).

However, there is a pressing need to understand in greater detail


the many factors that contribute to ribosome function and the
regulation of translation on a global scale. Thus, we set out to
analyze in a nonbiased, high-throughput manner the numerous
factors that coordinate ribosome function and mRNA translation.
Here, we present an overview of the riboproteome, as characterized
by analysis of several different cell lines and different cellular
contexts.

HYPOTHESIS
They hypothesize that the process of active
translation within the cell is regulated by a
multitude of proteins that can interact with
either the ribosome itself,
the mRNAs that are being actively translated,
or
proteins that may have the capacity to interact
with both the ribosome and mRNA.

WHAT DID THEY DO???


In order to characterize the components that
constitute the actively translating ribosome (i.e.,
the riboproteome),
They applied a mass spectrometry approach to
quantitatively evaluate the protein components
that are differentially associated with translation
in different cellular contexts, while also allow for
a comprehensive overview of the proteins that
make up the riboproteome.

EXPERIMENTAL PROCEDURES

SILAC Labeling and Mass Spectrometry


Metabolic labeling of prostate cancer cell lines
(PC3, PPC1, Du145, RWPE1, and PWR1E) and
MEFs was carried out using either normal
arginine and lysine or heavier isotopic variants of
the two amino acids (L-lysine 2HCL [U-13C6], Larginine HCL [U-13C6, U-N15N4]) (Ong et al.,
2002) using Invitrogens SILAC-FLEX Media kits.

EXPERIMENT
SILAC (stable isotope
labeling by amino acids in
cell culture) media to
incorporate amino acids for
light (Lys0 C13; Arg0 N14) or
heavy (Lys6 C13; Arg10 N15)
labeling of proteins,
achieving a labeling
efficiency of greater than
95%

Importantly, an initial comparison


between polysomes derived from Du145
heavy- and light-labeled cells revealed
that all quantified
proteins showed an average Log2 (H/L)
ratio of around 0 (226 quantified proteins;
mean 0.0029, SD 0.1866

demonstrating that differences observed


between cell
lines do not arise from variations in
sample preparation and confirming
both reliability as well as reproducibility
of the technique.

Polysome Isolation and Analysis

Standard scatterplots with normalized Log2 (H/L) ratios/Log10 Intensities (Normal versus Cancer
n = 3, left panel; Cancer versus Cancer n = 3, middle panel;
PPC1 DMSO versus PP242, n = 3, right panel) highlighting the distribution of quantified proteins
in each screen (cutoff values for enriched proteins was 2 SDs (2s)
from the mean, dashed red lines). Proteins of interest in either experimental setting are
highlighted.

Bioinformatics Analysis of the


Riboproteome

To identify functional gene sets enriched in the


riboproteome genes, they uploaded the
riboproteome genes to Ingenuity Pathway
Analysis
(http://www.ingenuity.com)
and
identified the top biological functions gene sets
and canonical pathways gene sets enriched in
the riboproteome gene set.
To identify KEGG pathways specifically enriched
in the subset of riboproteome genes they
uploaded the five of five experiments gene list to
DAVID and used the one of five experiments
gene list as background.
Gene ontology analysis was carried out using the
online DAVID bioinformatics resource tool.

TCGA-Based Analyses of Riboproteome


Genomic Alterations across Human Cancers

Western Blot Analysis


Western blot analysis from
pooled polysomal
fractions validating ribosomeassociated proteins
from ribosomes of PC3, PPC1,
Du145, RWPE1
and PWR1E cells. For this
analysis, polyribosomes
have been isolated from all cell
lines and fractions
have been pooled to obtain
subunits

DISCUSSION
First, by cross-referencing data from independent SILAC
riboproteomic experiments showed that data set is highly
enriched in factors that relate directly to the ribosome, to
translational initiation and elongation, and to pathways that
are known to regulate and control translation.
Second, the data sets indicate that the diversity within the
riboproteome itself may have the capacity to categorize cell
types and tissues and, importantly, may specifically contribute
to regulation of gene expression within a given cellular
compartment.

DISCUSSION
Third, by examining globally how the riboproteome may be
altered in diseases such as human cancer, we have made
further unexpected observations. We find that riboproteomic
components display frequent copy-number amplifications in
human cancer, whereas genomic losses within the
riboproteome are significantly less than that for
nonriboproteomic genes.
Fourth, in addition to characterizing the riboproteome
landscape in various cell types, we identified and validated a
number of proteins previously not known to be associated
with actively translating ribosomes
Last, our data demonstrate that the cancer riboproteome can
be pharmacologically modulated for therapy on the basis of
this molecular knowledge.

Highlights
Mass spectrometry identifies differential
riboproteome components in cancer cells
Riboproteomic genes are frequent targets of
genetic amplification in cancer
Riboproteomics
identifies
previously
unrecognized ribosome-associated proteins

THANK YOU