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For small RNAs that are usually shorter than 300 nucleotides, the biogenesis of snRNAs and snRNPs is surprisingly complicated (Note that in many yeast species, including S. pombe

and S. cerevisiae, U2 snRNA can be over 1kb due to an hypervariable insertion in the middle of the RNA). Assembly of most of the known spliceosomal snRNPs (U1, U2, U4, U5, U11, U12 and U4atac) and U7 snRNP can be divided into three phases, the first nuclear phase, the cytoplasmic phase and the second nuclear phase (Figure 1.8). The assembly of U6 and U6atac snRNPs occurs entirely inside the nucleus (Hamm and Mattaj, 1989; Vankanet al., 1990; Terns

et al., 1993; Boelenset al., 1995; Panteet al., 1997; Spilleret al., 2007).

After pol II type snRNAs are transcribed by pol II in the nucleus they are first m7G capped and then their 3’ ends processed by the integrator complex (Figure 1.8A). After pre- liminary processing, snRNAs are bound by the cap binding complex (CBC), Ars2 and Phax (phosphorylated adapter for snRNA export) (Ohno et al., 2000; Hallais et al., 2013). Certain snRNP-specific proteins associate with the snRNAs prior to export, for example, U1A, while others are assembled onto snRNPs in the cytoplasm or after reimport into the nucleus (Terns

et al., 1993; Kambach and Mattaj, 1994). The pre-export complex transits through the Cajal body and is exported to the cytoplasm with the help of Crm1 and Ran-GTP (Suzuki et al., 2010; Ohnoet al., 2000).

The Cajal body, initially discovered by Santiago Ramon y Cajal over 100 years ago, also known as the nucleolar accessory body or coiled body, is a unique subnuclear RNP granule present in many proliferative cells and neurons (see review in Gall (2000); Morriset al.(2008)). The Cajal body has been implicated in assembly and maturation of many RNP complexes, including the snRNPs, RNA polymerase, telomerase etc. (Darzacq et al., 2002). Cajal bodies are molecularly defined by the presence of coilin, and in most cases are associated with actively transcribing snRNA genes (Matera, 1999). In addition to Coilin and snRNPs, Cajal bodies also contain the SMN complex, snoRNPs (small nucleolar RNPs) and scaRNPs (small Cajal-body specific RNPs, including the telomerase complex). In some cell types (e.g. HeLa cells), SMN and some of its associated proteins form snRNP-free granules tightly associated with Cajal bodies, and are called gems (for Gemini of the Cajal bodies) (Carvalho et al., 1999; Matera, 1999). Histone processing factors such as U7 snRNP, FLASH etc. often form another kind of nuclear RNP granule at the histone loci called the histone locus body (HLB). HLB and Cajal bodies sometimes are very close to each other, or completely overlap (for review see Nizami

et al.(2010); Materaet al.(2009)). Both the outbound and inbound routes of snRNP transport go through the Cajal bodies, however, it is not entirely clear how Cajal bodies affect snRNP assembly. The widely accepted theory is that concentrating factors in a small volume accelerates chemical reactions, which in this case, results in more efficient snRNP assembly.

Figure 1.8: The biogenesis pathway of spliceosomal snRNPs(excluding U6 and U6atac, which are assembled through a different pathway localized in the nucleus). (A). The nuclear phase of snRNP assembly, from snRNA transcription to the assembly of pre-export and export complexes. (B). The cytoplasmic phase of snRNP assembly, where SMN assembles Sm proteins onto snRNAs, Tgs1 hypermethylates the m7G cap. After re-import, snRNPs are assembled into spliceosomes. CBC: cap binding complex. Phax: phosphorylated adapter for RNA ex- port. CRM1: chromosome region maintenance 1, also known as exporting or Xpo1. Tgs1: trimethylguanosine synthase 1. SMN: survival of motor neuron. SMNc: SMN complex. SPN: snurportin. Adapted from Matera and Wang 2014.

assembly ensue (Figure 1.8B), and these steps are catalyzed and coordinated by the SMN complex (SMNc) (Fischeret al., 1997; Massenet et al., 2002; Pellizzoniet al., 2002). The major proteins assembled onto the snRNAs are the Sm proteins. Three of the seven canonical Sm proteins, as mentioned above, are symmetrically dimethylated at the arginine residues (sDMA) in the RG boxes (Brahms et al., 2001). sDMA modification is required for efficient assembly of snRNPs by the SMN complex in human cells, but not in Drosophila cells. The methylation of Sm proteins is catalyzed mainly by the type II protein arginine methyltransferase (PRMTs) PRMT5, together with other proteins, pICln and WDR77/MEP50.

The SMN complex consists of multiple copies of SMN, Gemin2-8 and Unrip. Mutations and loss of the human SMN1 gene are known to cause a severe human disease, SMA (Spinal Muscular Atrophy). The SMN protein can oligomerize to form the scaffold for the whole SMN complex that contains other proteins. The best-studied functions of the SMN complex is in the assembly of the snRNPs. Recent crystallographic studies have started to reveal certain details of the assembly pathway. Gemin2, a conserved member of the SMN complex binds directly to five of the seven Sm proteins, the SmD1-SmD2-SmF-SmE-SmG pentamer and holds them in a semistable state for subsequent snRNA loading and ring closure (Zhang et al., 2011). The chaperon protein pICln, which is a component of the PRMT5 complex, mimics SmB-SmD3 dimer structure in vivo that stabilizes the pentamer before Gemin2 binding (Grimm et al., 2013; Chari et al., 2008). The Tudor domain of SMN contains an Sm fold, and is thought to also have a SmB-SmD3 mimetic role during Sm core assembly (Grimmet al., 2013).

After Sm proteins are assembled onto snRNAs, the m7G cap is hypermethylated to TMG by Tgs1, which is recruited by the SMN complex. Sm core assembly and TMG capping are two important signals for snRNP import back into the nuclei. snRNP import is mainly mediated by the snRNP import adapter Snurportin, which binds the TMG cap directly. Importin beta (Moleskin in flies) are the import acceptors for the Snurportin-snRNP complex. Interestingly, in addition to the nuclear Cajal bodies where many steps of snRNP assembly take place, cytoplasmic RNP granules call U bodies, have also been discovered that are related to snRNP assembly (Liu and Gall, 2007). However, little is known about which steps are organized in U bodies, and whether U bodies actually facilitate snRNP assembly (see Chapter 5 for more

results and discussion).

After snRNPs are imported into the nucleus together with the SMN complex, they localize temporarily to the Cajal bodies, where SMN complex dissociates from snRNPs, more snRNP- specific proteins are assembled, and box C/D and box H/ACA scaRNPs guide snRNA 2’-O- methylation and pseudouridylation respectively. snRNAs are extensively modified, and some of these modifications are required for splicing activity (for review see Karijolich and Yu (2010)).

Figure 1.9: A simple diagram of U2-type splicing. The splicing process rearranges the RNA-protein interactions in the spliceosome in a highly ordered manner to facilitate the the formation of the catalytic center of the spliceosome. In the first step, U1 and U2 snRNPs form base pairing interactions with the 5’ splice site (5’ss) and the intronic branch point sequence (BPS), respectively. U2 binding exposes the the branch point adenosine, indicated by the letter A. The first step forms the pre-spliceosome complex A. Subsequently, U4/U6.U5 tri-snRNP is recruited to the complex A to form the pre-catalytic spliceosome complex B. In complex B, U2 and U6 forms base pairing interactions, releasing U4 from the U4/U6 base paired di- snRNP, while U5 snRNP invariant loop base pairs with sequences in the 5’ exon. The 5’ end of U6 snRNA then base pairs with the 5’ss to release U1 snRNP from the spliceosome. After U1 and U4 are displaced, complex B* is formed. The complex B* then undergoes extensive rearrangement to bring 5’ss and the branch point adenosine close to each other and facilitate the first trans-esterification reaction. Further rearrangements occur to facilitate the second trans-esterification reaction. The center of U6 forms an intra-molecular stem-loop (U6-ISL), which is necessary for catalyzing the trans-esterification reactions. (Adapted from Matera and Wang 2014.)