Potential Benefits in Inequality? The Protective Role of U1 snRNP
Pre-mRNA in eukaryotes is spliced by the spliceosome, an RNA-protein complex, in which U1, U2, U4, U5 and U6 snRNPs are equal components. However, U1 is more abundant than other snRNPs in cells. U1 snRNP has been found to functions other than splicing, namely in protecting pre-mRNA from premature cleavage and polyadenylation. This protective role may account for its increased levels within cells.
In eukaryotic cells, pre-mRNA undergoes extensive post-transcriptional modifications to become mature mRNA. The modifications to pre-mRNA include 5’ end capping, 3’ end cleavage and polyadenylation, and the splicing of introns (Gu and Lima, 2005). The spliceosome is a large RNA-protein complex, which removes introns from pre-mRNA. Key components of the spliceosome are small nuclear ribonucleproteins (snRNPs). U1, U2, U4, U5 and U6 snRNPs help to form the major (U2-type) spliceosome. Functionally, snRNPs recognise and base pair to sequences that define introns in pre-mRNA (Wahl et al. 2009).
The snRNPs form the spliceosome in equal stoichiometry. Despite this, cellular concentrations of the snRNPs are unequal. In particular, U1 snRNPs are much more abundant than other snRNPs. U1 snRNP commonly recognises and defines 5’ splice sites in pre-mRNA (Berg et al. 2010). However, more recent work has shown that U1 snRNP has functions other than splicing, and this may account for the increased cellular levels.
To decipher the functions of U1 snRNP, a study by Kaida et al. designed antisense morpholino oligonucleotides (AMO) to reduce the level of functional U1 snRNP within HeLa human cells. The U1 AMO binds to and covers the 5’ end of U1 snRNA, preventing it from binding to 5’ splice sites on pre-mRNA. An RNase H protection assay was run to confirm U1 AMO binding to U1 snRNP. Increasing the concentration of U1 AMO reduced the cleavage of U1 snRNA, as U1 AMO protected it from cleavage, showing that it had bound and inactivated U1 snRNP both in vivo and in vitro.
Cells were transfected with U1 AMO, and then compared to a control transfected with scrambled U1 AMO. It was seen that more introns were present in the U1 AMO cell transcripts than in the control, showing that splicing was inhibited, and so that U1 is critical to spliceosome activity.
U1 AMO transfected cells were also compared to cells transfected with spliceostatin A (SSA), which inhibits splicing. As splicing was inhibited in both sets of cells, it would be expected that the same intron pattern would be seen, but this was not so. It was observed that in U1 AMO cells, introns tended to terminate early, often within the first 3-5 kb, and that in these transcripts, a polyadenylation sequence that is not encoded for by the genome was found at the 3’ end. The polyadenylation sequence was a consequence of a potential polyadenylation sequence (PAS) nearby.
The differences between U1 AMO cell...