Mechanism of Snorna Acting on the Serotonin Receptor

The mechanism used by snoRNPs to change splice site selection is not obvious, as snoRNPs are mainly located in the nucleolus and splicing takes place in the nucleo-plasma. However, snoRNAs are generated in the nucleoplasma during the splicing reaction and intron release. They can therefore contact pre-mRNA during their generation. In addition, snoRNAs share some proteins with the splicing machinery. For example, the 15.5 K protein that was originally identified as part of a the C/D box snoRNP complex where it binds to a conserved kink turn binds also to a similar structure in the U4 snRNA where it interacts with the splicing factor hPrp31 (Liu et al. 2007). This raises the possibility that the 15.5 K protein bound to snoRNPs interferes with the U4/U6 rearrangement during the splicing reaction by interacting with hPrp31.

Insight into the mechanism came from experiments that analyzed the RNAs from a single HBII-52 expression unit by RNase protection analysis, which directly quantifies the expressed RNAs. The data indicated that the HBII-52 expression unit generates several RNAs. Mutation studies showed that these shorter RNAs are only made when their precursor snoRNA contains intact C and D boxes. This indicates that they are most likely generated by further processing of the snoRNA and were therefore termed psnoRNAs (for processed snoRNAs) (Kishore et al. 2010). The main product of the 48 HBII-52 expressing units that are missing in PWS is therefore not a C/D box snoRNA but a psnoRNA that lacks several nucleotides at the ends. This shorter version lacks the stem of the snoRNA that is crucial for the assembly of a functional snoRNPs but still contains the antisense box needed for targeting to pre-mRNA. In addition to this form, three other shorter RNAs (60 to 37 nt) could be detected. The psnoRNAs were present in the nucleoplasma, where they could interact with pre-mRNA. The analysis of the protein composition showed that the RNAs associate with hnRNPs commonly implicated in splice site regulation but not with the known structural C/D box snoRNA proteins or the 2'-0-methylase (Kishore et al. 2010). This strongly suggest that HBII-52 has a role different from C/D box snoRNAs that function in 2'-O-methylation of RNA.

As the major HBII-52 psnoRNA form still contains the antisense box that targets the serotonin receptor exon Vb sequence, it is likely that this RNA form brings processing factors to this exon, similar to a bifunctional oligonucleotide. Studies of miRNAs, Dscam selector RNA or U1 snRNAs showed that RNA:RNA interactions can tolerate multiple mismatches towards their targets. This indicates that HBII-52 could also regulate other splicing events.

A bioinformatic analysis predicted about 220 alternative exons that have evolutionary conserved sites that exhibit limited complementarity to the antisense box. Five of these exons were regulated by HBII-52 expression (Kishore et al. 2010). In each of the identified exons there were three mismatches between the 18 nt antisense element and the target RNA, which is reminiscent of U1, where the majority of 5' splice sites has a mismatch in two of the nine possible bases.

Together, these data indicate that HBII-52 derived RNAs promote exonVb inclusion either by recruiting other pre-mRNA processing factors to 5-HT2C pre-mRNA or by forming novel small RNAs that interfere with the splicing process.

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