View source for 15.5kD/Snu13/L7Ae protein

<StructureSection load='' size='450' side='right' caption='Structure of 15.5kD bound with a U4 snRNP fragment ([[1e7k]])' scene='Sandbox_503/Start_scene/2'>
== Structure of the protein homologues: 15.5kD, Snu13, and L7Ae ==
   
__TOC__
 
=Introduction=

The human protein 15.5kD and its yeast (Snu13p) and archaeal (L7Ae) homologues function in the processing of pre-ribosomal RNA as part of the box [http://en.wikipedia.org/wiki/Small_nucleolar_RNA#C.2FD_box C/D] and [http://en.wikipedia.org/wiki/Small_nucleolar_RNA#H.2FACA_box H/ACA] small ribonucleoprotein particle (sRNP – [http://en.wikipedia.org/wiki/Archaea archaea]) or small nucleolar ribonucleoprotein particle (snoRNP – [http://en.wikipedia.org/wiki/Eukarya eukarya]) nucleotide modification complexes  (s(no)RNPs)<ref name ="gagnon">PMCID:PMC2802039</ref><ref name ="dobbyn">PMID:17631273</ref>.  In addition, 15.5kD and Snu13p function in U4 [http://en.wikipedia.org/wiki/SnRNP small nuclear ribonucleoprotein particle] (snRNP) spliceosomal biogenesis<ref name ="dobbyn"/>.  The capability to function in dual roles lies in the ability to recognize a [[Kink-turn motif|helix-bulge-helix (kink-turn) RNA motif]] that is present in the different RNPs<ref name ="oruganti">PMID:15963469</ref>
  
A variation of the [[Kink-turn motif|kink-turn motif]], known as the kink-loop motif, can be found in the C/D and H/ACA RNAs <ref name ="gagnon"/>.  Interestingly, the eukaryotic proteins and their archaeal homologue do not interact with the different motifs in the same manner, even though share a conserved sequence similarity <ref name ="oruganti"/>.  For example, while L7Ae exhibits the same binding affinity for both the [[Kink-turn motif|kink-turn]] and kink-loop sRNA motifs, its eukaryotic homologues only bind specifically to the [[Kink-turn motif|kink-turn motif]] and discriminate against the kink-loop motif <ref name ="oruganti"/><ref name ="gagnon"/>. 
 
Solved structures of the proteins include: 15.5kD in complex with a U4 snRNA fragment [[1E7K]], 15.5kD in complex with hPrp31 and a U4 snRNA fragment [[2OZB]], Snu13p without RNA [[1ZWZ]], [http://en.wikipedia.org/wiki/Archaeoglobus Archaeoglobus fulgidus] L7Ae-box C/D with RNA [[1RLG]], [http://en.wikipedia.org/wiki/Methanococcus_jannaschii Methanococcus jannaschii] L7Ae-H/ACA with RNA [[1RA4]], and [http://en.wikipedia.org/wiki/Pyrococcus Pyrococcus abyssi] L7Ae without RNA [[1PXW]].
  
=Role in pre-ribosomal RNA processing=
 
[[Ribosomes]] consist of both RNA and protein, and are designated large ribonucleprotein (RNP) particles.  Each ribosome contains two subunits (60S and 40S), four ribosomal RNAs (5S, 5.8S, 18S, and 25/28S rRNA), and approximately 75 associated proteins <ref name ="venema">PMID:10690410</ref>.  The processing of the pre-rRNAs requires a complex set of posttranscriptional modification steps after [http://en.wikipedia.org/wiki/Transcription_(genetics) transcription] <ref name ="venema"/>.  One such step involves extensive processing through pseudouridylation and 2’-O-ribose methylation at sites specified by various [http://en.wikipedia.org/wiki/Small_nucleolar_RNA s(no)RNAs] (C/D box s(no)RNAs specify 2’-O-ribose methylation and H/ACA s(no)RNA specify pseudouridylation) and associated proteins to form s(no)RNPs <ref name ="venema"/><ref name ="m-g">PMID:12810916</ref>.  Specifically, the 5’ region of U3 s(no)RNA containing C’/D and B/C box pairs interacts with 5’-ETS and 17S/18S areas of the pre-rRNA<ref name ="m-g"/>.  U3 also binds a set of proteins to form the U3 s(no)RNP complex <ref name ="gagnon"/>.
 
Snu13p/15.5kD/L7Ae interacts with U3 s(no)RNA through a kink-turn RNA motif <ref name ="venema"/>.   The protein initiates box C/D assembly by binding the kink-turn of the C/D RNAs <ref name ="gagnon"/>.  Once the s(no)RNP is fully assembled the RNA regions bind to complementary regions in target pre-rRNA.  This is followed by catalysis of the methyl transferase reaction by the associated proteins <ref name ="gagnon"/>.
  
=Role in pre-messenger RNA splicing=

The processing of [http://en.wikipedia.org/wiki/Pre-mRNA pre-mRNA] takes place through the use of a large dynamic machine known as the spliceosome, through which [http://en.wikipedia.org/wiki/Intron introns] are removed and [http://en.wikipedia.org/wiki/Exon exons] are spliced together to create a mature [http://en.wikipedia.org/wiki/MRNA mRNA]<ref name ="architecture of"/><ref name ="structure and function">PMID:19000813</ref>.  The spliceosome is comprised of five [http://en.wikipedia.org/wiki/SnRNA snRNA] molecules (snRNAs U1, U2, U4, U5, and U6) and over one hundred associated proteins<ref name ="architecture of"/><ref name ="structure and function"/>.  Assembly of the spliceosome is thought to take place in a stepwise manner around the pre-mRNA transcript<ref name ="architecture of"/><ref name ="structure and function"/>.  The first step involves recognition of the 5’ splice site by U1 snRNP, followed by recognition of the branch point sequence by U2 snRNP<ref name ="architecture of"/><ref name ="structure and function"/>.  From this point the tri-snNRP consisting of U4/U6•U5<ref name ="architecture of"/><ref name ="structure and function"/> together with the five snRNPs form the precatalytic spliceosome, which must undergo a series of changes before it can actively splice<ref name ="structural evidence">PMID:19525970</ref><ref name ="structure and function"/><ref name ="common design">PMID:19525970</ref><ref name ="architecture of">PMID:22471593</ref>.

U4 snRNA has a 5’ stem-loop containing a kink-turn that has been shown to interact with 15.5kD <ref name ="vidovic">PMID:11163207</ref>.  There is evidence to suggest that 15.5kD plays a role in late stage spliceosomal assembly, prior to splicing catalysis <ref name ="vidovic"/>.  In addition, it may be involved in binding other proteins that have been found to indirectly associate with U4 snRNA such as 61k (Prp31p in yeast), as well as the 20/60/90k complex which interacts with the U4/U6 duplex <ref name ="vidovic"/>.  The homologues for 60k and 90k in yeast are Prp4p and Prp3p respectively; there is no yeast homologue for 20k.

=Structure of 15.5kD in complex with U4 snRNA fragment=


__NOTOC__

=Overall structure=

15.5kD exhibits a globular domain structure, characterized by an α-β-α fold <ref name ="dobbyn"/>. The partial U4 snRNA oligonucleotide makes contacts with the protein at a pocket through nucleotide U31, which is located in the internal loop. The portion of [http://en.wikipedia.org/wiki/Oligonucleotide oligonucleotide] not contacting the protein, folds into two [http://en.wikipedia.org/wiki/Double_helix double helices] meeting to form a loop at the junction, where both are capped by a [http://en.wikipedia.org/wiki/Purine purine].  The RNA fold is stabilized through multiple hydrogen bond interactions and base stacking.  The overall structure of the protein and RNA is such that there is little interaction between the RNA and protein, leaving much of the proteins surface area exposed for interactions with other components in the U4 snRNP <ref name ="vidovic"/>. 
  
=15.5kD structure= 

15.5kD is 128 amino acids long and folds into a compact α-β-α sandwich, which is the most common family of protein folds, and resembles the L30 ribosomal protein.  The <scene name='Sandbox_503/B-sheet/4'>central</scene> [http://en.wikipedia.org/wiki/Beta_sheet β-sheet]contains four β-strands, one parallel and three antiparallel, and are ordered <scene name='Sandbox_503/B1/1'>β1</scene>, <scene name='Sandbox_503/B1-b4/1'>β4</scene>, <scene name='Sandbox_503/B1-b4-b2/1'>β2</scene>,  and <scene name='Sandbox_503/B1-b4-b2-b3/1'>β3</scene>.  To one side of the β-sheet [http://en.wikipedia.org/wiki/Alpha_helix α helices] <scene name='Sandbox_503/A1-a4-a5/1'>α1, α4, and α5</scene> are closely packed, and on the other side are <scene name='Sandbox_503/A2-a3/1'>α2 and α3</scene>.  The residues 63-66 of <scene name='Sandbox_503/310_helix/3'>α3</scene> form a 310 helix that is important in RNA binding; helix α2 also contains residues important for binding<ref name ="vidovic"/>. 

=U4 snRNA fragment structure=

The U4 snRNA fragment contains nucleotides <scene name='Sandbox_503/Rna_start/1'>26-47</scene> of the full length 5’stem loop.  The oligonucleotide forms into two distorted A-form RNA stems (stem 1 and stem 2) that are connected by an asymmetric 5+2 internal loop (<scene name='Sandbox_503/Rna_3_nucs/1'>2 nucleotides</scene> originate from the 3’ strand and <scene name='Sandbox_503/5_strand_nucs/1'>5 nucleotides</scene> originate from the 5’ strand).  The internal loop has a complex fold, where <scene name='Sandbox_503/G-a_bp_nucs/1'>four of its nucleotides</scene> from sequential G-A base pairs, and the other three are left unpaired.  Of the unpaired nucleotides <scene name='Sandbox_503/U31/1'>(U31)</scene> is flipped out, and the other two <scene name='Sandbox_503/A29_a30/1'>(A30 and A29)</scene> act as purine caps by stacking onto A44 of stem two, and the G45-C28 base pair of stem 1, respectively.  Between two of the G-A base pairs G32-A44 and G43-A33, the helix is overwound and causes cross stand stacking of the two adenines <scene name='Sandbox_503/A33_a44/1'>(A33 and A44)</scene>, leaving the guanines displaced.  One of the unpaired adenines <scene name='Sandbox_503/A33_a44_a30/1'>(A30)</scene> also participates in the cross stacking.  The overall structure represents the kink-turn motif<ref name ="vidovic"/>.

In addition to base stacking and pairing interactions that help stabilize the RNA structure, there is a network of [http://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] interactions that also contribute.  These predominantly involve ribose 2’OH groups and nitrogen or phosphate atoms.  The 2’OH of A44, A29, A33, U31, G32, and G43 hydrogen bond, or are within hydrogen bonding distance, of A30(N6), A44(N1), G45(N3), A30(P), G43(N2), and A44(P) respectively<ref name ="vidovic"/>.
  
=Protein-RNA interactions=

The residues of 15.5kD that play a main role in RNA binding, through interactions with the 5+2 internal loop, include those located in α2, α4, β1, and loops β1-α2, β2-α3, and α4-β4.  There are four major interactions; the first involves the flipped out U31 nucleotide; the second, the sequential G-A base pairs; the third, the unpaired and stacked adenines; and the fourth, the electrostatic interactions<ref name ="vidovic"/>.
 
====The flipped out U31 nucleotide====

As previously mentioned the residue U31 is flipped out, which makes it an optimal site for RNA-protein interactions.  It is located in pocket formed by the residues <scene name='Sandbox_503/61_65_86_100_u31_rna/1'>Glu61, Ile65, Lys86, and Ile100</scene>.  Multiple hydrogen bonds and Vander walls interactions are formed between U31 and the four residues.  The O4 and 3-imino group of U31 form a hydrogen bond with the main chain amide and main chain oxygen of Glu61, respectively.  O4 of U31 hydrogen bonds to the amino group of Lys86, and the U31 phosphate hydrogen bonds to the main chain amide of Ile 100.  In addition, the base of U31 is in Vander walls contact with the hydrophobic regions of the Ile65, Ile100, and Lys 86.  U31 also forms a hydrogen bond with a residue not found in the pocket through its phosphate to the main chain oxygen of Ala39<ref name ="vidovic"/>.

====The G-A base pairs====

Of the G-A base pairs G32-A44 and G43-A33, bases G32 and G43 interact with residues Asn40, Glu41, and Lys44, found in α2 and loop β2-α1, through their exposed atoms in the major groove. Atoms within hydrogen bonding distance include N1, N2, and O6 of G32 to the carboxylate group of Glu41, N7 and O6 of G43 to the ɛ-amino group of Lys44, O6 of G32 to the main chain amide of Asn40, and N7 of G32 to the ND2 of Asn40.  The adenines (A33 and A44) that base pair with the guanines (G32 and G43) do not form close interactions with the protein themselves<ref name ="vidovic"/>.
  
====The unpaired and stacked adenines====

The adenines that do not form base pair interactions (A29 and A30) participate in hydrophobic interactions with 15.5kD.  This takes place through their side regions which are not in contact with the base pairs of stem 1 (A29) and stem two (A30).  A29 packs with Arg97 in the α4β4 loop, and A30 packs with Val95 in loop α4-β4 and Lys37 on loop β1-α2<ref name ="vidovic"/>.
  
====The electrostatic interactions====

The RNA carries with it a negative charge due to the phosphate backbone.  This charge is stabilized by several basic residues in the 15.5kD protein.  The negative charges of phosphates C42 and A29 are stabilized by residues Lys44 and Agr97 respectively.   Residues Arg36, Arg48, and Lys37 are in close proximity to the RNA backbone (7-8Å) and help contribute to the electrostatic state of the RNA-protein complex<ref name ="vidovic"/>. 

=Structure comparison between 15.5kD, Snu13p, and L7Ae homologues=
 
Structurally, Snu13p and 15.5kD are more similar than either to L7Ae; however, they exhibit different binding affinities to cognate RNAs<ref name ="m-g"/><ref name ="oruganti"/>.  The eukaryotic proteins exhibit very specific binding (ie. will only bind to RNA with the kink-turn motif), whereas their archaeal homologue does not (ie. will bind to RNA exhibiting either the kink-turn or kink-loop motifs)<ref name ="m-g"/><ref name ="oruganti"/>.  Differences in structure between the archaeal and eukaryotic proteins lie in the α2-β2 loop and the β4-α6 loop which do not directly bind DNA, however have been shown to contribute to the structural integrity of the RNA binding elements<ref name ="oruganti"/>.  In the eukaryotic proteins there is the addition of two amino acids that creates further hydrogen bonding between β2 and β4, which in turn may provide further stabilization to α2<ref name ="oruganti"/>.  The N-terminus also carries an area of structural differentiation; preceding α1 in L7Ae is a random coil, whereas in 15.5kD and Snu13p there is a β-strand which may participate in further stabilization of the protein<ref name ="oruganti"/>.  Overall, the structures are very similar, such that the small differences do not seem likely to contribute to their differential binding.
  
The structure itself may not the most important aspect when comparing the homologues, rather the amino acid composition.  There are five amino acids located at the RNA binding region that are conserved within each of archaea and eukarya, however vary between the two.  One such amino acid lies towards the N-terminal side of the RNA binding region, in L7Ae it is Lys26 (Methanocaldococcus jannashii), and in 15.5kD it is Gln34.  Towards the C-terminal side of the RNA binding region located in loop 9 lie the four remaining residues Leu-Glu-Aal-Ala (L7Ae) and <scene name='Sandbox_503/Valserargpro/2'>Val95-Ser96-Arg97-Pro98 </scene>(15.5kD).  It is the difference between these amino acids that allow L7Ae to bind the kink-loop motif<ref name ="m-g"/>. 
</StructureSection>
=Additional Resources=

*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1e7k CRYSTAL STRUCTURE OF THE SPLICEOSOMAL 15.5KD PROTEIN BOUND TO A U4 SNRNA FRAGMENT, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=2ozb Structure of a human Prp31-15.5K-U4 snRNA complex, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1zwz Structural comparison of Yeast snoRNP and splicesomal protein snu13p with its homologs, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1rlg Molecular basis of Box C/D RNA-protein interaction: co-crystal structure of the Archaeal sRNP intiation complex, in the RCSB Protein Data Bank]
*[http://www.rcsb.org/pdb/explore/explore.do?structureId=1pxw Crystal structure of L7Ae sRNP core protein from Pyrococcus abyssii, in the RCSB Protein Data Bank] 
 
=References=
<References/>