Of Poly-A’s and rRNAs:

Polyadenylation in Artemia
franciscana 16s rRNA

Adult A. franciscana

By Preethi Jothi
July 2005
 Abstract
• We analyzed two clones, 21DE3 and 21PJ2, from an
Artemia franciscana cDNA library.
• We BLASTed both the sequences and found that both
sequences had a significant homology with 16S
mitochondrial rRNA of various Artemia species, as
well as Parartemia serventyi.
• Since both sequences matched same product, we
BLASTed both clones and found that the sequences
overlapped in 186 bases.
• We noticed that both sequences had relatively long
poly(A) tails, which were not present in the original
mitochondrial genome. Through further research we
discovered that the poly(A) tail has an important
role in the stability of the rRNA.
Introduction
We sequenced clones from the A. franciscana cDNA
library.
- Characteristics of Artemia:
•Live in hypersaline environments
•Under stressful conditions, the blastula forms a
dormant cyst
•In dormant cyst-form, can survive without oxygen
for years
- Applications of Research:
•Produce preservation and longer shelf-life in human
organ-transplants through suspended animation
techniques
•Develop taxonomy and evolutionary relationships of
organisms
Methods
•Isolated mRNAs of Artemia using Oligo-T column.
•Inserted mRNAs into vector.
•Cloned inserts through bacterial asexual
reproduction.
•Isolated plasmid by miniprepping.
•Cut plasmid into insert and vector through use of
EcoRI enzyme.
•Ran gels to determine lengths of inserts, then
sequenced the clones with the longest inserts.
•Edited sequences.
•Researched properties of gene product.
 Data (Decision to analyze 21DE-3 and 21PJ-2)
Results of gel electrophoresis

What does this mean?

- Uncut is purified plasmid.
- Cut is the purified plasmid subjected to the restriction enzyme EcoRI.
- The “1Kb Ladder Size Standard” tells us the length of each of the 1KB Ladder’s bands.

Other Observations:
- 21PJ-2 has two bands in the insert; perhaps the insert has an EcoRI site within it, resulting
in the enzyme cutting it.
- 21PJ-2 has a longer insert than 21DE-3; note that even when cut, the two bands of insert
are still longer than 21DE-3’s insert. (21PJ-2’s two bands are ~lengths 700 and 250 Kb, for a
total of ~950 Kb long. 21DE-3’s insert is only ~450 Kb long.)
 Results
Results of Sequence Alignment Assessment (BLAST 2 seq) between 21DE-3
and 21PJ-2
- Found that 21DE-3’s and 21PJ-2’s
sequences seemed highly similar while
editing.
- Ran a BLAST 2 seq align on both
sequences to assess similarity; found
that 21DE-3 was 94% similar to 21PJ-2.
- Closer examination of 21DE-3
revealed that the few errors were
caused by erroneous reading of the
sequence, and that it was actually
100% similar.
- Both sequences had a poly(A) tail at
the end.
- Also discovered that 21PJ-2 was the
cleaner sequence, and continued for
another 291 bases. Decided to use
21PJ-2 for further analysis.
 Results
Results of BLASTn (Comparing 21PJ-2’s nucleotide sequence to those on
NCBI’s Nucleotide Database)
- Found the closest results to be to A.
franciscana’s complete mitochondrial DNA and
Artemia’s mitochondrial 16S ribosomal RNA.
- Other matches included A. monica’s 16S
ribosomal RNA; partial sequences of A.
franciscana’s, A. parthenogenetica’s, A.
sinica’s, A. urmiana’s, A. tunisiana’s, and A
parsimilis’s 16S ribosomal RNA gene from the
mitochondrion.
- Concluded that the sequence must be 16S
rRNA. We found this quite unusual; we used
the Oligo-T column to isolate mRNA from other
genetic material, and yet we still obtained
rRNA, leading us to believe that the sequence
was polyadenylated. Stranger still, the same
rRNA sequence was obtained twice from two
totally separate clones (21PJ-2 and 21DE-3).
 Results
Results of Sequence Alignment Assessment (BLAST 2 seq) Between 21PJ-2
and A. franciscana’s complete mitochondrial DNA

- We decided to assess where the

similarities occurred between 21PJ-2
and A. franciscana’s complete
mitochondrial DNA, since not all of
21PJ-2 matched.

- Discovered that 467 bases of our

sequence matched to the
mitochondrial genome. The other 11
bases that did not match were a
poly(A) tail.

- There were two mismatches. We

determined that these may have been
due to variations in the population of
Artemia.
 Results
Comparison of 21PJ-2 to the mitochondrial genome
- Although there were several series of A’s within our sequence that matched
with the mitochondrial genome, there were still 10 bases in our sequence--
10 consecutive A’s--that did not align.
- We decided to compare it once more to the mitochondrial DNA, except this
time looking for the sequence of the mitochondrial sequence where we had
A’s. (See figure.)

- We found that where we had a series of A’s followed by immediate

termination, the mitochondrial DNA had a different sequence and continued
on for several thousand more bases.
- One possible interpretation of these results is that the poly(A) tail was
added after transcription--which is quite surprising, considering that for
years we believed that rRNA was never polyadenylated.
 16S rRNA in the Formation of the 70S Initiation Complex

Results
The function of 16S rRNA

- Out of curiosity, we decided to research the

function of 16S rRNA.
- Discovered that 16S rRNA is a part of the 30S
ribosomal P site and is vital to the formation of
the 70S initiation complex.
- The 30S P site serves several key roles in
translation (binds initiator tRNA during formation
of 30S initiation complex; binds anticodon stem-
loop of peptidyl-tRNA during elongation; maintains
the translational reading frame when the A site is
1. Binding on IF1 & IF3 to an empty 70S
unoccupied). ribosome dissociates it into 50S and 30S
subunits.
- 70S initiation complex required to begin 2. a. 5' region of mRNA binds to free 30S
interaction with tRNA, and 16S rRNA required to ribsomal subunit, IF3 is released.
b. Initiator fMet-tRNAfMet carried by
create 70S initiation complex. IF2-GTP binds to P site of 30S ribsomal
subunit.
- Therefore, 16S rRNA is required to support 3. Free 50S ribosomal subunit binds, IF1 &
IF2-GDP + Pi are released.
protein synthesis in living cells.
 Results
The function of polyadenylation in rRNA
- Next, we researched the role of polyadenylation in rRNA.
- We found that polyadenylation of rRNA without edits speeds up rRNA
degradation, and polyadenylation with edits greatly inhibits degradation.
However, the edits alone do not inhibit degradation, as edits alone result in
degradation (albeit slower than polyadenylation alone). (See figure.)

Therefore, polyadenylation either greatly reduces rRNA stability or greatly enhances

Discussion
Since Artemia franciscana 16S mitochondrial rRNA has already
been sequenced, what is the significance of our clone?
We have found the first cDNA match to 16S rRNA in Artemia.

Why was there rRNA in the cDNA library?

The rRNA sequence had a poly(A) tail, which attached to the oligo-T
column.

Were there A’s in the mitochondrial genome?

No. So this means one of two things:
1. Because of variations in the population, maybe some
individuals whose DNA hasn’t been sequenced yet have a string
of A’s at that locus.
2. The poly(A) tail was added after transcription.
 Post-transcriptional polyadenylation of mitochondrial
rRNA has recently been found in other organisms and
may play a role in rRNA stability.
Applications for Further Research
- Due to the relative ease required to isolate the 16S
rRNA sequence combined with our discovery of poly(A)
tails in the sequence, it is a good model for research in
polyadenylation in Artemia’s rRNA sequences.

- In Drosophila, the frequency of rRNA contamination in

the cDNA library is high. Therefore, it is important to
determine how to prevent rRNA contamination of the
cDNA library in order to make the process more
efficient. The more knowledge we have concerning
these polyadenylated sequences, the more likely it is
that a solution to this problem can be found.
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