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C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-Omethylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA-RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously "silent" exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes.alternative splicing | gene regulation | snoRNAs | pre-mRNA processing S mall nucleolar RNAs (snoRNAs) are 60-to 300-nt-long noncoding RNAs that accumulate in the nucleolus. Based on conserved sequence elements, snoRNAs are classified as C/D box small nucleolar RNAs (SNORDs) or H/ACA box snoRNAs (SNORAs). SNORDs contain sequence elements termed C (RUGAUGA) and D (CUGA) boxes, usually present in duplicates (C′ and D′ boxes), and up to two antisense boxes that hybridize to the target RNA (1). In humans, SNORDs are usually derived from introns. After the splicing reaction, introns are excised as lariats, which are then opened by the debranching enzyme and subsequently degraded. Intronic SNORDs escape this degradation by forming a protein complex that consists of non-histone chromosome protein 2-like 1 (NHP2L1, 15.5K, SNU13), nucleolar protein 5A (NOP56), nucleolar protein 5 (NOP58), and fibrillarin (2-4). The SNORD protein complex forms through the entry of the snoRNA and fibrillarin to a complex containing NHP2L1, NOP58, and at least five assembly factors (5). The SNORD acts as a scaffold for the final protein complex formation and also controls recognition of other RNAs using the antisense boxes. The antisense boxes recognize sequences in rRNA, resulting in the fifth nucleotide upstream of the D or D′ box being 2′-O-methylated by fibrillarin (1). Structural studies indicate that the active form of SNORDs is dimeric (6).The conserved overall structure of SNORDs allows the identification of their putative target RNA binding sites. However, numerous SNORDs without obvious target RNAs have been identified (7-10) and are termed "orphan snoRNAs." Genome-wide deep sequencing experiments identified shorter but stable SNORD fragments that were found in all species tested, ranging from mammals to the protozoan Giardia lamblia (11) and EpsteinBarr virus (12). Fragments longer than 27 nt generated by SNORDs will ...

Almost every protein-coding gene undergoes pre-mRNA splicing, and the majority of these pre-mRNAs are alternatively spliced. Alternative exon usage is regulated by the transient formation of protein complexes on the pre-mRNA that typically contain heterogeneous nuclear ribonucleoproteins (hnRNPs). Here we characterize hnRNP G, a member of the hnRNP class of proteins. We show that hnRNP G is a nuclear protein that is expressed in different concentrations in various tissues and that interacts with other splicing regulatory proteins. hnRNP G is part of the supraspliceosome, where it regulates alternative splice site selection in a concentrationdependent manner. Its action on alternative exons can occur without a functional RNA-recognition motif by binding to other splicing regulatory proteins. The RNA-recognition motif of hnRNP G binds to a loose consensus sequence containing a CC(A/C) motif, and hnRNP G preferentially regulates alternative exons where this motif is clustered in close proximity. The X-chromosomally encoded hnRNP G regulates different RNAs than its Y-chromosomal paralogue RNA-binding motif protein, Y-linked (RBMY), suggesting that differences in alternative splicing, evoked by the sexspecific expression of hnRNP G and RBMY, could contribute to molecular sex differences in mammals.All protein-coding genes undergo pre-mRNA processing, and the large majority of these genes are alternatively spliced (1). Alternative exons can change many functional aspects of mRNAs and their encoded proteins. The best understood functions are stop codons or frameshifts that are introduced by 20 -35% of alternative exons, which often destine the altered mRNA to nonsense-mediated decay. Examples described in the literature show that alternative splicing regulates the binding properties, intracellular localization, enzymatic activity, protein stability, and post-translational modifications of a large number of proteins (reviewed in Ref.2). Thus, it appears that alternative pre-mRNA processing is a key mechanism regulating the gene expression of complex organisms by generating multiple mRNA isoforms, which encode functionally diverse proteins. Despite its importance, the exact mechanisms governing splice site selection are still poorly understood. In vertebrate systems, protein complexes assemble transiently on exons, and their interaction with the splicing machinery as well as RNA-RNA interactions between spliceosomal proteins and pre-mRNA determine whether an exon is included or skipped (reviewed in Refs. 3 and 4).When isolated from nuclei of mammalian cells, RNA polymerase II transcripts are found assembled in large ribonucleoprotein 21-MDa complexes, the supraspliceosome, composed of all five spliceosomal small nuclear ribonucleoproteins as well as additional proteins. The entire repertoire of nuclear pre-mRNAs, independent of their length or number of introns, is individually found assembled in supraspliceosomes (reviewed in Ref. 5). Structural studies revealed that the supraspliceosome is composed of four substructure...

Splicing of pre-mRNA occurs in a multicomponent macromolecular machine--the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 angstroms. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.

Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory pre-mRNA splicing event is fundamental in development and cancer. It occurs within a mega-Dalton multicomponent machine composed of RNA and proteins, which undergoes dynamic changes in RNA-RNA, RNA-protein, and protein-protein interactions during the splicing reaction. Recent years have seen progress in functional and structural analyses of the splicing machine and its subcomponents, and this review is focused on structural aspects of the pre-mRNA splicing machine and their mechanistic implications on the splicing of multi-intronic pre-mRNAs. It brings together, in a comparative manner, structural information on spliceosomes and their intermediates in the stepwise assembly process in vitro, and on the preformed supraspliceosomes, which are isolated from living cell nuclei, with a view of portraying a consistent picture.