Tuesday, May 12, 2009

Small RNAs Get Smaller

Tiny RNAs recently joined the growing list of non-coding RNA (ncRNA) molecules [1]. Their absolute function is not understood, but they are possibly a new class of ncRNA and appear to be most associated with transcription of highly expressed genes in human, chickens and Drosophila and possibly others.

This was the conclusion of work published in the May issue of Nature Genetics. Remember when all we had to worry about was the central dogma? DNA was transcribed into RNA and RNA was translated into protein. Life was so simple.

Not really. Even as the first genetic code was being elucidated [2], the possibility of uncovering the second code, that translates nucleic acid sequences into protein sequences was being contemplated [3]. Translating RNA into protein required other kinds of RNA that became known as ribosomal (rRNA) and transfer RNA (tRNA). The RNA between DNA and protein became messenger (m)RNA. In the late seventies, introns were discovered [4,5] and soon to follow were small nuclear (sn)RNAs and “snurps” (small nuclear riboproteins). The snRNAs were further characterized as small nucleolar (snoRNA) and Cajal body-specific (scaRNA) RNAs, and a class of new molecules were investigated for their involvement in mRNA splicing.

As the mechanisms for splicing were being worked out, researchers were able to prove that RNA could also be an enzyme [6]. In this case, the intron is the enzyme responsible for splicing itself out to create the mature mRNA. At the same time, another group discovered that the catalytic unit of RNAase-P, an enzyme involved in converting precursor tRNAs into active tRNAs, is also RNA [7]. Indeed, later work revealed that rRNA in the large ribosome subunit catalyzes the peptidyl transferase reaction to join amino acids together to build proteins [8]. Not only does the central dogma require a multitude of RNA molecules to transcribe DNA into RNA and translate RNA into protein, but the RNA molecules are responsible for carrying the information needed to make proteins and supplying the enzymatic activity to do the work!

What else does RNA do?

More than we can imagine. Starting with the discovery that double stranded RNA (dsRNA) could inhibit gene expression by turning on RNA interference (RNAi) pathways [9], new RNAs were identified, micro (miRNA) and small interfering (siRNA), as essential to the RNAi pathway. miRNA and siRNA were the early members of what would become a large and growing class of RNAs now referred to as non-coding RNAs (ncRNAs).

The ncRNAs represent a next frontier in RNA research and understanding gene expression. Some ncRNAs are large, like lincRNAs (large intervening non-coding RNAs) [10], but most are small between 18 and 31 nt. Within in the small ncRNA group are piwi-interacting (piRNA), repeat associated small interfering (rasiRNA), small temporal (stRNA), and now transcription initiation (tiRNA) RNA. I like tiny RNA.

Tiny, or tiRNAs, were discovered by Next Generation Sequencing (NGS) studies. RNA libraries were prepared from specific size fractions of capped messages. The resulting libraries were sequenced on the Roche FLX Genome Sequencing system and the data were aligned to human genome build 36.1 and compared to transcription start sites (TSS) defined by RefGene (NCBI). The authors reasoned the previous deep-sequencing studies missed these RNA molecules because they tend to be disregarded as low-abundance spurious, or degradation products. However, because they can be cloned, they must have a 5’ phosphate and, when aligned to genomc sequences, the NGS reads cluster in a non-random fashion around TSSs.

GeneSifter enables small RNA research

NGS makes it possible to explore the RNA world in new ways by designing experiments to capture small RNA molecules and sequence them in a massively parallel, high throughput format. However, both the experiments and data analysis are technically challenging. Fortunately GeneSifter Laboratory Edition (GSLE) and GeneSifter Analysis Edition (GSAE) can help. In GSLE you can use the software to track RNA preparation steps and record data at different points of the process. GSAE is accompanied with data analysis pipelines designed to filter artifacts and identify known small RNAs. Post alignment clustering reports, based on coverage in a genome, can be used to further refine results an discover new RNA species as well. Moreover, you can convert the clustering reports into lists of expression values for these RNAs and compare their expression between different samples, tissues, or experimental conditions.

1. Taft R.J., Glazov E.A., Cloonan N., et. al., 2009. Tiny RNAs associated with transcription start sites in animals. Nat Genet 41, 572-578.

2. Watson J.D. and Crick F.H.C. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 171, 737-738 (1953)

3. Crick F.H., Barnett L., Brenner S., Watts-Tobin R.J., 1961. General nature of the genetic code for proteins. Nature 192, 1227-1232.

4. Chow L.T., Roberts J.M., Lewis J.B., Broker T.R., 1977. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids. Cell 11, 819-836.

5. Berk A.J., Sharp P.A., 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12, 721-732.

6. Zaug A.J., Cech T.R., 1982. The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA. Nucleic Acids Res 10, 2823-2838.

7. Guerrier-Takada C., Gardiner K., Marsh T., Pace N., Altman S., 1983. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849-857.

8. Nissen P., Hansen J., Ban N., Moore P.B., Steitz T.A., 2000. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920-930.

9. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C., 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

10. Guttman M., Amit I., Garber M., French C., Lin M.F., Feldser D., Huarte M., Zuk O., Carey B.W., Cassady J.P., Cabili M.N., Jaenisch R., Mikkelsen T.S., Jacks T., Hacohen N., Bernstein B.E., Kellis M., Regev A., Rinn J.L., Lander E.S., 2009. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223-227.

Further Reading
ncRNA - http://nar.oxfordjournals.org/cgi/reprint/35/suppl_1/D178
snoRNA - http://en.wikipedia.org/wiki/SnoRNA
siRNA - http://en.wikipedia.org/wiki/SiRNA
miRNA - http://en.wikipedia.org/wiki/MicroRNA
piRNA - http://en.wikipedia.org/wiki/Piwi-interacting_RNA
rasiRNA - http://en.wikipedia.org/wiki/RasiRNA
stRNA - http://jcs.biologists.org/cgi/content/full/116/23/4689
Ribozymes - http://en.wikipedia.org/wiki/Ribozyme

miRBASE - http://microrna.sanger.ac.uk/sequences/
RNAdb - http://research.imb.uq.edu.au/rnadb/default.aspx