Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition

Scientific comments:

1) Pre-mRNA binding assays: For oligo 5ss-F, the sequence does not appear to be a consensus 5'-splice site. Thus, the competing oligos have a consensus splice site but the sequence differs by more than a single-nucleotide position. The authors need to explain why these sequences were used or repeat the assay using oligos with the proper sequence.

We found that a labelled consensus sequence oligonucleotide bound too tightly to be competed off by the weaker competitor oligonucleotides at concentrations that could be feasibly achieved in the experiment. Hence we used a labelled oligonucleotide with one mismatch (5SS-F). This does not influence the experiment's capacity to show whether one oligonucleotide competes better or worse than another.

2) The main text provides no details about how the crystal structures were determined, i.e. by molecular replacement.

We now make it clear in the main text that both structures were determined by molecular replacement with a sentence added to the end of the first paragraph of the Results section.

The rationale for using the U4 snRNP core domain as a model was not explained. Is it simply because the Sm proteins are shared?

It is now explained in Materials and methods section.

General comments:

Interactions are described extensively throughout this manuscript. However, specific interactions (i.e. amino acid residues and nucleotides) are usually not stated. If specific interactions are mentioned, they often are not labeled in the figures, making it difficult to understand the authors' interpretation of the structure. More details need to be added to the figure legends to accurately and precisely describe the interactions shown in the figures.

We have now labelled amino acid residues and nucleotide as requested in the figures. Chains are coloured consistently throughout the manuscript.

Abstract:

The resolution of the crystal structures (3.3 and 2.5 Ang) needs to be added. Also, “subdomains” is misleading because this term is not generally applied to the U1 snRNP. Perhaps the authors should use “synthetic substructures” instead.

The second sentence of the Abstract has been changed to “We present two crystal structures, at 2.5 Å and 3.3 Å resolution, of engineered U1 sub-structures which together reveal...” Although the term 'synthetic' was suggested to describe the sub-structures, we believe the term “engineered” already suffice to describe “something which was man-made”. Also, we now use the hyphenated term 'sub-structure', rather than 'substructure' throughout the text, because the latter only has the dictionary definition, 'an underlying or supporting structure', which is not the intended meaning.

Introduction:

The range of nucleotides in the invariant sequence is not mentioned. The authors should insert “(nts #-#)” to specify the nucleotides of interest?

We inserted (nts1-10).

The Introduction compares 5′SS recognition in yeast and humans. It appears the authors are trying to make the point that this work is focused on the conserved regions. To make this point more clear, perhaps the authors should begin by explaining how the yeast and human U1 snRNPs differ. Although “human” is used in the Title, the authors should reconfirm that all the components in this work are based on human nt sequences and proteins, correct?

p5: To address several points made about the Introduction, an extra sentence has been added to the 4th paragraph, comparing yeast and human U1 snRNP, and the nucleotide range has been inserted in the subsequent sentence.

“Yeast U1 snRNP, when compared to the human particle, contains a larger and more complex snRNA, which is associated with many protein factors (Prp39, Snu71, Prp40, Prp42, Nam8, Snu56, Urn1 and Prp5), which have no counterparts in human U1 snRNP (Will and Lührmann, 2011). However, despite these differences, the sequence of the 5'-single stranded region of U1 snRNA (nts 1-10) is invariant from yeast to human”.

The Introduction does not end with a short summary of the major findings of this work, leaving the reader unsatisfied. The last paragraph of the Introduction is a run-on paragraph. The authors should divide the paragraph into two independent paragraphs: one for the yeast-human comparison and one for the reported findings.

A short summary paragraph has been added to the end of the Introduction.

“The two crystals together reveal the structures of the substantial parts of U1 snRNP at high resolution and provide crucial insights into the mechanism of pre-mRNA recognition by U1 snRNP. In particular, we find that U1-C makes no base-specific contacts with the 5'SS sequence. Also, by measuring the intrinsic affinity of recombinant U1 snRNP for various 5’SS sequences we disentangle the role played by the U1 snRNP from the other complexities of 5’SS recognition, and assess the relative contributions of U1 snRNA and U1-C protein in light of our crystal structure.”

The Introduction talks about the role of U1 in splicing. However, there is no mention of its other important cellular function, telescripting.

An extra sentence has been added to the end of paragraph 1, mentioning the role of U1 snRNP in regulating cleavage and polyadenylation of mRNA (telescripting) with two references.

“U1 snRNP is also an important regulator of mRNA 3' end cleavage and polyadenylation (Almada et al., 2013; reviewed in Spraggon and Cartegni, 2013).”

Results:

The text mentions seven Sm proteins but Figure 1A labels only five Sm proteins.

We have now labelled all components in Figure 1A.

“The overall structure of the core domain is very similar to that of the U4 snRNP core domain.” Please include rmsd to support the descriptor “very similar”. Also, how do you know the similarity is not due to model bias?

We included rmsd in the text. The U4 snRNP structure has now been refined to Rfree of 22.4% and the U1 snRNP structure has been refined to Rfree of

25.5%. Hence model bias would not be an issue for ordered regions we discuss in this manuscript. We included rmsd of the two structures in the text.

Helix H0 and helix H are two examples of features being mentioned that are not shown/labeled.

It is shown in Figure 1A and we now refer to this figure in the text.

What are the “key residues”? The residues are also not identified in the figure legend. The reader must guess what residue is at the “equivalent position”. Where are loops L3 and L5?

The key residues in loops L3 and L5 are now identified in Figure 2–figure supplement 1 legend.

The authors talk about nucleotides yet the exception they list, SmD1, is a protein. This is confusing.

This has been changed to clarify that nucleotide G130 interacts differently with protein SmD1.

Binding is stabilized by salt bridges and hydrogen bonds – what residues and/or nucleotides participate?

We added “its binding to SmD2 is stabilized…” to clarify this statement.

Because U4 is referred to several times throughout this manuscript, a picture of U4 should be shown in the supplement, explaining what components constitute the “core”.

We added “which consists of seven Sm proteins and U4 snRNA when the U4 core snRNP first appears”.

The text specifically identifies strands and helices of importance but these secondary structures are not labeled in the figures (e.g. beta 4 strand in Figure 3D and 4A). There are other such omissions throughout the manuscript.

See Figure 3D. We do not consider it necessary to label the 3₁₀ helix of U1-70k in Figure 4A because this helix is defined as residues 6-12 in the text and the start and end residues (Pro6 and Leu12) are labeled in the figure. Furthermore, this region of the figure is already quite crowded.

Be quantitative about how much the R21Q mutation weakens binding.

We added “by about 10-fold” to this sentence.

Are pseudo-uridines present in both human and yeast snRNAs?

Pseudo-uridines are conserved at the 5' end of U1 snRNA of yeast and human. The following has been added to paragraph 4 of the Results section and a supporting reference has been added to References (see below).

“Pseudo-uridines at these positions are conserved from yeast to human U1 snRNA (Massenet et al., 1999).”

The “E complex” needs to be defined and its relevance explained for the general reader.

“At an early stage of spliceosomal assembly U1 snRNP binds to the 5’SS and U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) bind to the 3’ splice site and branch point resulting in the formation of E complex.” This has been inserted before the sentence to define the E complex.

“Conserved well” – what exactly is the percentage of sequence similarity?

“The first 215 residues of U1-70k are conserved well from yeast to human (48% sequence similarity), suggesting that this region has an evolutionarily conserved essential function”.

What is meant by the 11-nt loop effectively being a 5-nt loop? How does base stacking reduce loop length?

This is illustrated in Figure 5 A and C.

C33 and G34 are not labeled in Fig 5A. In Fig 5c, the side chains of R191 and K138 should be shown?

Labels for C33 and G34 and the side chains of R191 and K138 have been added to Figure 5A.

The filter binding assay is not adequately described in Materials and methods.

See Materials and methods, end of final section.

'Mechanism of 5'SS recognition', first paragraph: to be more accurate about U1 snRNP affinity for 5'SS RNA ±U1-C, the penultimate sentence has been altered.

“The affinity of U1 snRNP without U1-C (U1 snRNP( U1-C)) for the wild type 5’SS oligonucleotide increases by between three and four-fold on addition of U1-C (Figure 7A).”

Materials and methods:

1) Are all proteins and nt sequences based on human?

“All proteins are based on human sequences, unless otherwise indicated” was added as the second sentence in the Protein Preparation section and the sentence. “All RNAs are based on human sequences, unless otherwise indicated” was added to the RNA Preparation section.

2) Prior to the induction step, were cells grown at 15^°C?

The growth temperature prior to induction has been added.

“The cells were grown at 37°C in 2xTY medium with ampicillin (100 ∝g/ml) and, when A_{600 nm} = 0.4-0.8, protein expression was induced by addition of 0.5 mM IPTG and the culture was continued at 15°C overnight.”

3) What is the final storage buffer? Was glycerol not added?

The given method is correct; no glycerol was added. However, the complex was flash-frozen in liquid nitrogen before storage and an explanatory clause has been added.

“The U1-70kSmD1/SmD2 heterodimer was eluted by a NaCl gradient and peak fractions µM, were pooled, concentrated to ∼300 rapidly frozen in liquid nitrogen and stored at -80°C.”

4) No storage conditions are described. Was the protein never frozen?

Storage conditions have been added as follows:

“Peak fractions were concentrated by ultrafiltration, buffer exchanged into 20 mM Na.Hepes, 25 mM imidazole, 0.3 M NaCl, pH 7.5, rapidly frozen in liquid nitrogen and stored at -80°C.”

5) Steps are described for annealing full-length U1 snRNA but it is not stated to what it is being annealed. Do the authors mean folding rather than annealing? Also, is the annealing step performed in the absence of buffer?

The term annealing is commonly used to refer to the process of folding RNA by heating and subsequent cooling. We do not believe there is any ambiguity here. It is correct that the annealing was performed in the absence of buffer; optimal conditions were found empirically to be in 50 mM KCl.

End of final section: Added description of Filter-Binding Assay.

“In the filter-binding assay, a 5'SS RNA oligonucleotide (5'-³²P-CAAAGGUAAGAUGGA-3', 10 fmol), was mixed with various concentrations of U1 snRNP, with or without U1-C protein, in Filter Binding Buffer (FBB: 200mM KCl, 2 mM MgCl₂, 0.5 mM DTT,µg/mL10tRNA,mM Hepes, pH 7.5, 100 50µg/mL BSA) in a final volume of 40 µl and incubated at 22°C for 2 hours.

The binding reaction was passed, under vacuum, through a Schleicher & Schuell NC45, 25 mm diameter filter that had been pre-wetted with FBB. The filter was washed with 0.5 mL FBB and then dried. The proportion of radiolabeled oligonucleotide retained on the filter was determined by scintillation counting. The experiment was performed in triplicate.”

Figures & legends:

The descriptions in the figure legends are inadequate. Many of the figures suffer from the same problems. Please explain what is represented by colored objects and dashed lines in each figure legend. The colors in Figure 1–figure supplement 1 are not consistent with Figure 1 (e.g. SmF is brown in Figure 1 but green in the supplement). Please add more information about interactions between specific residues and nucleotides. Make sure key secondary structures, nucleotides and amino acids mentioned in the text are labeled in the figures, especially Figures 3 and 4.

Figure 1: For readers unfamiliar with the U1 snRNP, it would be helpful if there were a cartoon schematic for each crystal structure. Then, it would be easier to visualize the location of each component. Also, Figure 1 could show representative examples of the electron density for each structure. Then, Figure 2 could be deleted and replaced with Figure 2–figure supplement 1, which contains more substantial structural information.

Figure 1A: Labels for SmB and SmD1 were not put on this figure because these proteins are mostly concealed at the back of the Sm ring. Labels have now been added to indicate their position and color-coding. The positions of RNA Helix H and SmD2 helix H0 are now also indicated on the figure.

Figure 1 legend: This sentence has been added to part A to explain the labels H and H0 in panel A.

“Label H indicates U1 snRNA helix H; H₀ indicates the first alpha helix of SmD2 protein.”

Figure 1–figure supplement 1: What are the black, blue and red lines?

In response to the general comment, this figure has been re-colored to be properly consistent with Figure 1.

Figure 1–figure supplement 1 legend:

“The thinner lines below the bars represent the constructs used for crystallization: blue lines for sequences used in the U1A70kF fusion protein, red lines for sequences used in the 70kSmD1F fusion protein, and black lines indicate the extent of protein constructs used in minimal U1 crystallisation.”

Figure 2: Is this a stereoview?

First line now indicates that this is a stereoview.

“Stereoview showing binding of U1 snRNA at the central hole of the Sm protein assembly.”

Figure 2–figure supplement 1: The legend mentions electron density for A125 in panel A but no density is shown. Please add more details about panels B-H; the amino acid residues are important as well as the nucleotides.

This sentence has been moved into the Figure 2 legend from the legend for Figure 2–figure supplement 1 and altered slightly for clarity.

“Electron density for A125 and the phenol ring of SmF Y39, which stacks on it, is weak.”

Figure 2–figure supplement 1 legend: Has been re-written to address the comments about loops L3 and L5 and 'key residues' made regarding Results section.

Figure 4: What do the teal/pale pink colors represent?

The meaning of the teal and pale pink (fawn) nucleotides is explained.

“(B) U1-C forms hydrogen bonds with the sugar-phosphate backbone atoms but makes no contact with RNA bases. On the 5'SS strand, nucleotides are colored teal for exonic and fawn for intronic sequence. (C) Schematic representation of the 5'-splice site recognition. Red dotted lines, hydrogen bonds made by amino acid side chains of U1-C; blue dotted lines, hydrogen bonds made by main chain atoms of U1-C. The 5'SS nucleotides are color-coded as in panel B.”

Figure 7: A description of the plots is missing. What are “CPM” and “mP”? Do the error bars represent SD or SE? What are the Kd values extrapolated from the data in Figure 7A? The text mentions a 4-fold difference but doesn't state a numerical value with units. In panels C and E, WT data are missing.

Figure 7A legend: Made clear that the graph is of the results of a filter-binding experiment, added Kd values from filter-binding and defined 'CPM'.

“(A) Filter-binding results for U1 snRNP reconstituted with and without U1-C to [³²P]-labelled 5’splice site oligonucleotide. By curve fitting, the Kd with U1-C is 4.7 ± 0.8 nM and without U1-C is 15.8 ± 2.5 nM. CPM, counts per minute.”

Figure 7C legend: A sentence has been added to the end of this section of the legend defining 'mP' and explaining the error bars.

“In panels C-F, mP is an arbitrary unit of fluorescence polarization and error bars indicate standard error.”

Instead of WT, we use the +2C data to compare Figures 7C to 7D and Figures 7E to 7F; this prevents the figures from looking too crowded.

Tables:

In Table 2, is the sequence for -1C correct? It seems that the red U should be a C.

The sequence for -1C was indeed incorrect. This row has been amended. We have also removed rows -2U and +6A because these sequences weren't used in the experiments reported in this paper. A wild-type (WT) row has been added for the reader's reference.