Analysis of SINE Families B2, Dip, and Ves with Special Reference to Polyadenylation Signals and Transcription Terminators

Short Interspersed Elements (SINEs) are eukaryotic non-autonomous retrotransposons transcribed by RNA polymerase III (pol III). The 3′-terminus of many mammalian SINEs has a polyadenylation signal (AATAAA), pol III transcription terminator, and A-rich tail. The RNAs of such SINEs can be polyadenylated, which is unique for pol III transcripts. Here, B2 (mice and related rodents), Dip (jerboas), and Ves (vespertilionid bats) SINE families were thoroughly studied. They were divided into subfamilies reliably distinguished by relatively long indels. The age of SINE subfamilies can be estimated, which allows us to reconstruct their evolution. The youngest and most active variants of SINE subfamilies were given special attention. The shortest pol III transcription terminators are TCTTT (B2), TATTT (Ves and Dip), and the rarer TTTT. The last nucleotide of the terminator is often not transcribed; accordingly, the truncated terminator of its descendant becomes nonfunctional. The incidence of complete transcription of the TCTTT terminator is twice higher compared to TTTT and thus functional terminators are more likely preserved in daughter SINE copies. Young copies have long poly(A) tails; however, they gradually shorten in host generations. Unexpectedly, the tail shortening below A10 increases the incidence of terminator elongation by Ts thus restoring its efficiency. This process can be critical for the maintenance of SINE activity in the genome.

Mpe1 senses the polyadenylation signal in pre-mRNA to control cleavage and polyadenylation

Most eukaryotic messenger RNAs (mRNAs) are processed at their 3'-end by the cleavage and polyadenylation factor (CPF/CPSF). CPF mediates endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3'-end of the mature transcript. Activation of CPF is highly regulated to maintain fidelity of RNA processing. Here, using cryoEM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. This RNA-mediated link between the nuclease and polymerase modules promotes activation of the CPF endonuclease and controls polyadenylation. Mpe1 rearrangement is antagonized by another subunit, Cft2. In vivo, depletion of Mpe1 leads to widespread defects in transcription termination by RNA Polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in selecting the cleavage site, activating CPF and ensuring timely transcription termination.

Gene Editing Targeting the DUX4 Polyadenylation Signal: A Therapy for FSHD?

Facioscapulohumeral dystrophy (FSHD, OMIM: 158900, 158901) is the most common dystrophy in adults and so far, there is no treatment. Different loci of the disease have been characterized and they all lead to the aberrant expression of the DUX4 protein, which impairs the function of the muscle, ultimately leading to cell death. Here, we used gene editing to try to permanently shut down DUX4 expression by targeting its poly(A) sequence. We used transcription activator-like effector nucleases (TALEN) and CRISPR-Cas9 nucleases in vitro on FSHD myoblasts. More than 150 TOPO clones were sequenced and only indels were observed in 4%. Importantly, in 2 of them, the DUX4 poly(A) signal was eliminated at the genomic level but DUX4 mRNA was still produced thanks to the use of a non-canonical upstream poly(A) signal sequence. These experiments show that targeting DUX4 PAS at the genomic level might not be an appropriate gene editing strategy for FSHD therapy.