Birthing histone mRNAs by CSR-1 section.

As the building blocks of chromatin, histones need to be synthesized efficiently and synchronously with DNA replication. The mRNAs encoding the core histones undergo a distinct 30-end formation pathway that results in mature mRNAs ending in a stem loop instead of a polyadenylated tail. Considering that the factors involved in histone 30-end processing are well conserved across Eukarya, the absence of some of these components in C. elegans draws the question of how histone 30 ends are matured in worms. In this issue of The EMBO Journal, Avgousti et al (2012) present an answer that, surprisingly, involves RNAi pathway factors. Histone genes encode for pre-mRNAs that undergo maturation through a mechanism distinct from all other RNA Pol II transcripts by substituting polyadenylation with an alternative 30-end formation pathway (Marzluff et al, 2008). The 30-end of a histone pre-mRNA is marked by a short stemloop followed by a histone downstream element (HDE). The hairpin is bound by the stem loop binding protein (SLBP) and the HDE is recognized by the U7 small nuclear RNA (U7 snRNA) through base pairing interactions. Next, cleavage and polyadenylation specificity factor 73 (CPSF73) joins the complex and cleaves the pre-mRNA a few nucleotides downstream of the stem loop. SLBP remains associated with the processed histone mRNA and fulfills roles normally attributed to the polyA binding protein, such as promoting nuclear export and translation. Although the 30-ends of C. elegans histone mRNAs also terminate in stem loops instead of polyA tails, the U7 snRNA and its associated protein LSM11 appear to be missing in this species (Davila Lopez and Samuelsson, 2008). Additionally, worms lack an HDE-like sequence after the stem loop. These deficiencies prompt the question of how histone 30-ends are formed in C. elegans. One solution to this problem is presented in a new study, where RNAi-related factors regulate the formation of histone mRNA 30-ends (Avgousti et al, 2012). Instead of utilizing an HDE to position the cleavage complex, sequences downstream of the stem loop in histone pre-mRNAs were found to be complementary to endogenous small interfering RNAs (endo-siRNAs) (Figure 1). These endo-siRNAs are produced by the RNA-dependent RNA polymerase (RdRP) EGO-1 and associate with the Argonaute CSR-1 (Claycomb et al, 2009; Avgousti et al, 2012). In the absence of ego-1 activity, the interaction of CSR-1 with an in vitro synthesized RNA mimicking the 30-end region of the worm H2A pre-mRNA was lost, indicating that endo-siRNAs are required for CSR-1 to associate with this sequence of RNA. CSR-1, EGO-1, or another RNAi-related factor, the helicase DRH-3, were shown to be important for histone mRNA biogenesis in vivo as loss of any one of these factors resulted in decreased levels of histone mature mRNAs and proteins. Notably, histone pre-mRNAs were elevated in these conditions, supporting a novel role for RNAi factors in maturation of histone mRNAs. Another connection between the RNAi factors and histone mRNA biogenesis is the phenotypic similarities in worms depleted of csr-1 with those lacking cdl-1, which encodes the worm SLBP. Loss of cdl-1 results in histone deficiencies that are associated with chromosome segregation defects, sterility and lethality. CSR-1 was named for the chromosome segregation and RNAi defects observed upon loss of this factor, and similar abnormalities are detected in ego-1 and dhr-3 mutants (Yigit et al, 2006; Claycomb et al, 2009). Since decreased histone expression leads to embryonic lethality, transgenes designed to encode histone mRNAs that should be independent of the RNAi pathway factors were tested for rescue of this phenotype in worms depleted of csr-1 and ego-1.