Biology Reference
In-Depth Information
The amino acid carried out by the tRNA is then incorpo-
rated into the nascent polypeptide chain. Termination of
translation happens when the ribosome faces a stop codon
(usually UAA, UAG, or UGA) that induces the binding of
a release factor protein that prompts the disassembly of the
entire ribosome
transcripts is invariably the first step after the completion of
the sequence of a genome, since the vast majority of the
biology of a genome is initially inferred from the set of
genes predicted to be encoded in its sequence. To delineate
the reference transcriptome of a given genome, usually
a combination of high throughput RNA sequencing and
computational gene and transcript modeling is carried out.
In some selected genomes, such as that of human, full-
length sequencing of targeted RNA species may also be
attempted.
mRNA complex. After synthesis, proteins
fold and are transported to subcellular compartments where
they localize and exert their function.
In contrast to prokaryotes, in eukaryotes the presence of
the nuclear membrane leads to cellular compartmentaliza-
tion, where RNA processing in the nucleus is physically
separated from translation in the cytosol. Within the nucleus,
the different steps of RNAprocessing are also often thought as
separate processes occurring in a stepwise manner: tran-
scription, capping, polyadenylation and splicing. However,
there is increasing evidence of strong interconnections among
all of these processes. Indeed, on the one hand there appears to
be coordinated regulation of alternative splicing and alter-
native polyadenylation and cleavage [13] ; and on the other
hand splicing appears to occur essentially during transcription
[14] . These observations provide a natural explanation for
a number of recent observations that link chromatin structure
to splicing regulation [15] .
Some families of small non-coding RNA genes
(sncRNA) follow specific processing pathways, distinct
from those of long RNAs. They are usually transcribed as
precursors by variants of the RNA polymerase different
from those involved in the transcription of long RNAs; they
then undergo further processing. Ribosomal RNAs (rRNA)
are often transcribed as pre-rRNAs that contain one or more
rRNAs. The pre-rRNA is cleaved and modified at specific
sites by small nucleolus-restricted RNA species called
snoRNAs (small nucleolar RNAs). SnoRNAs themselves,
also involved in the processing of transfer RNAs (tRNAs)
and small nuclear RNAs (snRNAs), are usually encoded in
the introns of proteins involved in ribosome synthesis or
translation. In tRNAs, the 5 0 and 3 0 ends of the precursor
are removed to generate the mature RNA. Micro-RNAs
(miRNAs) are first transcribed as primary transcripts or pri-
miRNAs with a cap and poly(A) tail and processed to short,
70-nucleotide stem
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Experimental Methods
Random cDNA Cloning
A complementary DNA (cDNA) is a DNA sequence
synthesized by reverse transcription using an RNA mole-
cule as a template. cDNAs obtained from RNAs of interest
can be cloned in an appropriate vector, and the resulting
clones can be sequenced. Using oligo-dT probes that are
complementary to the poly(A) tail of long eukaryotic
RNAs, a cDNA library containing copies of many different
transcripts expressed in a given cell type or condition can
be created. Systematic in-depth sequencing of cDNA
libraries is likely the most powerful approach for the
identification of genes and transcripts. Traditionally, one
popular strategy is 'single-pass' sequencing of random
cDNA clones that results in the production of short partial
sequences identifying a specific transcript, known as
expressed sequence tags [16] (ESTs). In turn, ESTs can be
used to identify clones suitable for targeted full-length
sequencing, as in the Mammalian Gene Collection initia-
tive [17] . However, unless very large sequencing capacity is
available, the wide dynamic range of mRNA abundances in
the eukaryotic cell makes random sequencing of cDNA
clones inefficient for discovering relatively rare transcripts,
because highly abundant cDNAs will predominantly be
sequenced. In the absence of such sequencing capacity,
procedures that increase the likelihood of sampling rare or
tissue-specific transcripts, such as normalization and
subtraction, were developed [18,19] . These rely on dena-
turation of cDNA, rapid re-hybridization in the presence of
a 'driver' sample (the same sample in normalization;
a different one in subtraction), extraction of the double-
stranded portion of the sample, and finally sequencing of
a sample of the remaining single-stranded sequences. The
rapid re-hybridization step favors duplex formation of
abundant species, therefore the remaining single-stranded
sample is enriched for rare transcripts. This has been the
preferred approach during the last two decades for gener-
ating large EST collections used to delineate the reference
transcriptomes of many species. Normalization, however,
destroys all quantitative information. Therefore, while
random sequencing of cDNA clones can be successfully
loop structures known as pre-miRNAs
in the nucleus by the enzymes Drosha and Pasha. After being
exported to the cytosol, they are further processed to mature
miRNAs by the endonuclease Dicer, which also initiates the
formation of the RNA-induced silencing complex (RISC),
where the miRNA and its mRNA target interact.
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METHODS TO DETERMINE THE REFERENCE
TRANSCRIPTOME
We will use the term reference transcriptome to refer to the
set of all mature transcript RNA sequences encoded in
a given genome. Delineating this set of genes and
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