Small Nuclear Rnps (Snrnps) (Molecular Biology)

In addition to the short conserved sequences at the branch site and the 5′ and 3′ splice sites, RNA splicing of nuclear pre-mRNA is dependent on the activity of a large number of trans- acting factors. These can be divided into two classes: the snRNPs, evolutionarily conserved ribonucleoprotein particles, and non-snRNP protein splicing factors (reviewed in Refs. 1 and 2). These trans-acting factors assemble in an ordered fashion onto the pre-mRNA substrate to form the spliceosome, wherein the catalysis of splicing occurs. The spliceosomal snRNPs play a central role in the recognition and alignment of the pre-mRNA splice sites during spliceosome formation and are also involved in the catalytic steps of splicing (see Spliceosome). The major spliceosomal snRNPs, which are involved in the removal of U2-dependent introns (the most abundant type of nuclear pre-mRNA intron), include U1, U2, U5, and U4/U6, and are named according to their snRNA component(s). Due to their greater abundance (2*105 to 106 particles per cell, compared with 50 to 100 in yeast), the major spliceosomal snRNPs have been best characterized at the biochemical and structural level in higher eukaryotes and will thus be emphasized in the following paragraphs. In addition to U1, U2, U5, and U4/U6, a minor class of spliceosomal snRNPs involved in the excision of U12-dependent introns (which represent a small minority of nuclear pre-mRNA introns) has also been recently identified. These include the U11, U12, and U4atac/U6atac snRNPs, which, unlike the ubiquitous major snRNPs, appear to be lacking in some eukaryotes (eg, in the budding yeast S. cerevisiae) (3 5). As a consequence of their low abundance (estimated at 103 to 104 particles per cell) and more recent identification, the minor spliceosomal snRNPs are presently not well-characterized and thus will not be discussed in detail here.

1. snRNA

The major spliceosomal snRNPs consist of one snRNA molecule (or two in the case of U4/U6) complexed with a number of proteins. The U1, U2, U4, U5, and U6 snRNAs are characterized by their small size (164, 187, 145, 116, and 106 nucleotides, respectively in humans), metabolic stability, and high degree of sequence conservation. They also contain a large number of modified nucleotides, such as pseudouridine and 2′- O-methylated nucleosides, and, with the exception of U6, possess a unique 2,2,7-trimethylguanosine -cap structure. In addition to their sequence conservation, the secondary structures of the metazoan snRNAs are also highly conserved such that a consensus secondary structure model can be generated for each of them. The sequence and most probable secondary structures of the human U1, U2, U4, U5, and U6 snRNAs are shown schematically in Figure 1. The majority of the U4 and U6 snRNAs interact by extensive base pairing to form a characteristic Y-shaped structure and are present in this form within the U4/U6 snRNP. The most highly conserved sequences of these snRNAs are typically single-stranded regions that either base-pair with the pre-mRNA or other snRNAs during spliceosome assembly (eg, the 5′ end of U1) or serve as binding sites for snRNP proteins (eg, the so-called Sm site; see text below). With the exception of U4 and U6, the major spliceosomal snRNAs are somewhat larger in yeast (eg, U1 and U2 are 568 and 1175 nucleotides long, respectively), and they therefore exhibit different secondary structures. It should be noted that the structures of most of the spliceosomal snRNAs are not fixed, but rather undergo conformational changes during splicing that reflect the dynamic nature of the splicing process. A prime example is the U6 snRNA, which first base-pairs with U4 in the U4/U6 snRNP and then, after the incorporation of the U4/U6 snRNP into the spliceosome, dissociates from this complex and basepairs with the U2 snRNA and the pre-mRNA (see Spliceosome). The U4 snRNA and the 5′ half of the U2 snRNA also undergo significant conformational rearrangements during splicing (6). During splicing, the spliceosomal snRNAs engage in multiple base pairing interactions with other spliceosomal snRNAs and with the pre-mRNA. These RNA-RNA interactions play essential roles in the selection and favorable alignment of the 5′ and 3′ splice sites for splicing catalysis (see Spliceosome). Some of the structural features of the complex RNA network that is formed during spliceosome assembly mimic RNA structures that are essential for the autocatalytic splicing of group II pre-mRNA self-splicing introns. The spliceosomal snRNAs—in particular, U2, U5, and U6—thus appear to form, at least in part, the active sites of the spliceosome and take part directly in the catalysis of nuclear pre-mRNA splicing.

Figure 1. Sequence and secondary structure models of the human U1, U2, U5, and U4/U6 snRNAs. The consensus seco shown are the models of Guthrie and Patterson (15), and that of U2 was proposed by Ares and Igel (16). The conserved s of the common snRNP proteins) is underlined. The 5′ ends of the snRNAs possess a cap structure (2,2,7-trimethyl-guanc spliceosomal snRNAs (with the exception of U6, which possesses a monomethyl phosphate cap).

2. snRNP Proteins

The spliceosomal snRNAs are present in cells as discrete ribonucleoprotein complexes. In human cells, the U1 and U2 snRNAs are organized as a 12 S and 17 S particle, respectively. U4/U6 and U5 snRNAs can be isolated as individual 12 S and 20 S particles, respectively, but they also associate with one another to generate a 25 S [U5.U4/U6] tri-snRNP complex, and they are integrated into the spliceosome as such. The protein composition of the major spliceosomal snRNPs has been best characterized in HeLa Cellsand is summarized in Table 1. Proteins associated with the U1, U2, U5, and U4/U6 snRNPs fall into two classes. The first class consists of the so-called common or Sm proteins, denoted B ‘ , B, D1, D2, D3, E, F, and G, which are tightly associated with all snRNP particles. The Sm proteins play an important role in the biogenesis of the snRNPs and are essential for their import into the nucleus (see text below). The second class is comprised of the particle-specific proteins, which associate with a particular snRNP particle or complex. These proteins exhibit a wide range of binding affinities, and their association with an snRNP particle is thus dependent on the ionic strength of the particle’s environment. In addition to the eight common snRNP proteins, the metazoan U1 snRNA is associated with three proteins, designated 70K, A, and C. Under physiological conditions, the U2 snRNA is complexed with 12 U2-specific proteins, whose molecular weights range from 33 to 160 kDa (Table 1). With 16 specific proteins and two sets of common proteins, the 25 S [U4/U6.U5] tri-snRNP possesses the most complex protein composition of the snRNPs (Table 1). The vast majority of proteins identified in isolated HeLa snRNPs are also present in the spliceosome (7), and thus most spliceosomal proteins have an snRNP origin. The majority of the human snRNP proteins have now been cloned and sequenced, and several have been shown to perform essential functions during spliceosome assembly and the catalytic steps of splicing (see Spliceosome). For example, snRNP proteins (eg, U1-70K, U5-220 kD, and the U2-specific proteins comprising SF3a and SF3b) are involved in protein-protein interactions and protein-RNA interactions that facilitate the interactions of the U1, U2, and U4/U6.U5 tri-snRNPs with the pre-mRNA during spliceosome assembly. Furthermore, several snRNP proteins (eg, the 20 kD, 100 kD, 116 kD, and 200 kD U4/U6.U5 tri-snRNP proteins) appear to possess enzymatic activities, such as RNA duplex unwinding or protein isomerization activity, that potentially drive the many conformational changes that occur in the spliceosomal RNA and/or protein networks. In this way, snRNP proteins make important contributions to the assembly of the active sites responsible for the catalysis of splicing. However, whether snRNP proteins contribute directly to catalysis is currently an open question.

Table 1. Protein Composition of Human snRNPs®

C	22	/		y U1-C
A	34	/		Mud1p
70K	70	/		Snp1p
	28.5
	31
	33
	35
	92
	60			Prp9p
	66			Prp11p
	110			Prp21p
	53			Hsh49p
	120
	150			Cus1p
	160
	15
	40
	65
	100			Prp28p
	102
	110
	116			Snu114p
	200			Snu246p
	220			Prp8p
	15.5		+
	20		+
	60		+	Prp4p
	90		+	Prp3p
	27
	61
	63

^a The presence of a given protein in a particular snRNP is indicated by the various symbols. The 25 S [U4/U6.U5] tri-snRNP complex contains two sets of common proteins (E, F, G, D1, D2, D3, B, B’). Tri-snRNP proteins that also associate with U4/U6 snRNPs are indicated by a ( ), and those also present in U5 snRNPs are marked with a solid diamond. Proteins associated solely with the 25 S [U4/U6.U5] are marked with a solid square. SF3a is composed of the 60, 66, and 110 kDa U2-specific proteins, and SF3b consists of the 53, 120, 150, and 160 kDa U2-specific proteins. Additional information (including references) about the yeast S. cerevisiae common proteins and U1 snRNP specific proteins are reported in Ref. 8 and for the Prp proteins in Ref. 2. Snu246 is also referred to as Brr2p (17), Slt22p (18), and Rss1p (19) The identification of Prp3p and Prp4p as homologues of the U4/U6 90 and 60 kDa proteins is described in Refs. 12 and 20. Hsh49p and Cus1p are described in Refs. 21 and 22, respectively. (Courtesy of Claudia Schneider.)

Homologues of some of the HeLa U1, U2, U5, and U4/U6 snRNP proteins have also been identified either genetically or biochemically in yeast (summarized in Table 1). Significant progress has recently been made in the biochemical characterization of snRNPs from the yeast S. cerevisiae. This has complemented, as well as extended, the somewhat limited information obtained through yeast genetic techniques. Biochemical characterization of the yeast U1 snRNP has demonstrated that it possesses a significantly more complex protein composition than the metazoan U1 snRNP, containing not only homologues of the U1-70K, A and C proteins, but also six additional yeast-specific proteins (8). Some of these additional proteins appear to mediate the bridging of the 5′ and 3′ splice sites during the earliest stages of spliceosome formation in yeast. Although the number of snRNP protein homologues that have been characterized at the molecular level in yeast is presently limited, there are striking examples of both structural and functional conservation between yeast snRNP proteins and their counterparts in evolutionarily distant organisms. This not only underscores the functional importance of the snRNP proteins in nuclear pre-mRNA splicing, but also confirms that many aspects of the splicing process are conserved between higher and lower eukaryotes.

3. snRNP Biosynthesis and Structure

The snRNPs undergo a complex pathway of biogenesis that involves their shuttling between the nucleus and cytoplasm. Subsequent to their transcription in the nucleus, the major spliceosomal snRNAs, with the exception of U6, migrate to the cytoplasm. In the cytoplasm, some of the nucleosides of the snRNAs are modified (primarily 2 ‘ OH methylation and pseudouridylation) and their 5′ m G cap is hypermethylated to a 2,2,7-trimethylguanosine (m3G) cap. Cap hypermethylation is dependent upon the formation of an snRNP core structure. Core snRNPs are formed by the association of the common snRNP proteins (B ‘ , B, D3, D2, D1, E, F, G) with the Sm site, an evolutionarily conserved structural motif found in the U1, U2, U4, and U5 snRNAs (see Fig. 1). The Sm proteins form distinct heteromeric complexes (eg, E/F/G or B/D3) that interact with the Sm site in an ordered manner. Subsequent to core snRNP formation and cap hypermethylation, the snRNPs are translocated to the nucleus by an active, receptor-mediated process. The nuclear import pathway of the spliceosomal snRNPs is distinct from that of karyophilic proteins. snRNPs possess a bipartite nuclear localization signal that is comprised of the m3G cap and the snRNP core structure. At least one of the receptors responsible for snRNP import, Snurportin1, which specifically recognizes the m3G cap, has recently been described (9). SnRNP assembly is completed in the nucleus, where most of the particle-specific proteins are thought to associate with the snRNPs.

Although the vast majority of spliceosomal snRNP proteins have already been identified in humans, and a growing number are rapidly being identified in yeast, significantly less information about structural aspects (ie, RNA-protein and protein-protein interactions) of the spliceosomal snRNPs is currently available. Because the snRNPs serve as spliceosomal subunits, information regarding their higher-order structure may provide much needed information about the three-dimensional structure of the spliceosome. Information about the general morphology of both the mammalian and yeast spliceosomal snRNPs has been obtained through electron microscopy. More detailed information about RNA-protein and protein-protein interactions within the snRNPs is, however, limited. The molecular characterization of snRNP proteins has revealed that they possess a variety of interesting structural motifs, which include, among others, RNA-binding and protein-interaction domains (eg, RRMs, RS, and WD-40 domains). The best-characterized interactions within the snRNPs are those formed between snRNP proteins containing an RNA-binding domain (eg, U1-70K, U1-A, U2-B ‘ ‘ ) and their cognate snRNA. The atomic structure of interactions between the U1-A protein and U1 snRNA, and between the U2-B’ ‘/U2-A ‘ proteins and the U2 snRNA, have been obtained by X-ray crystallography (10, 11). Well-characterized protein-protein interactions include those formed between U2-snRNP-specific proteins comprising the splicing factors SF3a and SF3b, as well as those among the Sm proteins. More recently, a number of novel snRNP protein-protein interactions have been reported that involve proteins associated with the [U4/U6.U5] tri-snRNP complex (12-14).