The fission yeast methyl phosphate capping enzyme Bmc1 guides 2'-O-methylation of the U6 snRNA.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5' γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2'-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2'-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5' γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2'-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2'-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.

The evolution of pre-mRNA splicing and its machinery revealed by reduced extremophilic red algae.

The Cyanidiales are a group of mostly thermophilic and acidophilic red algae that thrive near volcanic vents. Despite their phylogenetic relationship, the reduced genomes of Cyanidioschyzon merolae and Galdieria sulphuraria are strikingly different with respect to pre-mRNA splicing, a ubiquitous eukaryotic feature. Introns are rare and spliceosomal machinery is extremely reduced in C. merolae, in contrast to G. sulphuraria. Previous studies also revealed divergent spliceosomes in the mesophilic red alga Porphyridium purpureum and the red algal derived plastid of Guillardia theta (Cryptophyta), along with unusually high levels of unspliced transcripts. To further examine the evolution of splicing in red algae, we compared C. merolae and G. sulphuraria, investigating splicing levels, intron position, intron sequence features, and the composition of the spliceosome. In addition to identifying 11 additional introns in C. merolae, our transcriptomic analysis also revealed typical eukaryotic splicing in G. sulphuraria, whereas most transcripts in C. merolae remain unspliced. The distribution of intron positions within their host genes was examined to provide insight into patterns of intron loss in red algae. We observed increasing variability of 5' splice sites and branch donor regions with increasing intron richness. We also found these relationships to be connected to reductions in and losses of corresponding parts of the spliceosome. Our findings highlight patterns of intron and spliceosome evolution in related red algae under the pressures of genome reduction.

Identification of Alternative Polyadenylation in Cyanidioschyzon merolae Through Long-Read Sequencing of mRNA.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga, Cyanidioschyzon merolae, to study the biology of polyadenylation in an organism highly diverged from humans and yeast. C. merolae is an acidothermophilic alga that lives in volcanic hot springs. It has a highly reduced genome (16.5 Mbp) and has lost all but 27 of its introns and much of its splicing machinery, suggesting that it has been under substantial pressure to simplify its RNA processing pathways. We used long-read sequencing to assess the key features of C. merolae mRNAs, including splicing status and polyadenylation cleavage site (PAS) usage. Splicing appears to be less efficient in C. merolae compared with yeast, flies, and mammalian cells. A high proportion of transcripts (63%) have at least two distinct PAS's, and 34% appear to utilize three or more sites. The apparent polyadenylation signal UAAA is used in more than 90% of cases, in cells grown in both rich media or limiting nitrogen. Our documentation of APA for the first time in this non-model organism highlights its conservation and likely biological importance of this regulatory step in gene expression.