Preethi Jothi - Research Project At Rutgers University

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 stemloop of peptidyl-tRNA during elongation; maintains the translational reading frame when the A site is unoccupied). - 70S initiation complex required to begin interaction with tRNA, and 16S rRNA required to create 70S initiation complex. - Therefore, 16S rRNA is required to support protein synthesis in living cells.

1. Binding on IF1 & IF3 to an empty 70S ribosome dissociates it into 50S and 30S subunits. 2. a. 5' region of mRNA binds to free 30S ribsomal subunit, IF3 is released. b. Initiator fMet-tRNAfMet carried by IF2-GTP binds to P site of 30S ribsomal subunit. 3. Free 50S ribosomal subunit binds, IF1 & IF2-GDP + Pi are released.

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.

Bibliography Benkel, Duschesnay, Boer, Genest, and Hickey. Mitochondrial large ribosomal RNA: an abundant polyadenylated sequence in Drosophila. Nucleic Acids Res. 1988 October 25; 16(20): 9880. Kao, Chia-Ying and Read, Laurie K. Opposing Effects of Polyadenylation on the Stability of Edited and Unedited Mitochondrial RNAs in Trypanosoma brucei. Mot Cell Biol. 2005 March; 25(5): 1634-1644. LaCava, John. et at. "RNA Degradation by Exosome Is Promoted by a Nuclear Polyadenylation Complex." Cell 121, 713-724. June 2005. Luecke, Hartmut Hudel. "Formation of the 70S initiation complex." Hartmut "Hudel" Luecke at UC Irvine. UC Irvine. 20 July 2005      . Noller, HF, L Hoang, and Fredrick K. "The 30S ribosomal P site: a function of 16S rRNA." FEBS letters (Feb. 2005): 855-8. 20 July 2005 .